Erythrocyte Membrane Antigens Research Articles

A newly characterized malaria antigen on erythrocyte and merozoite surfaces induces parasite inhibitory antibodies.

We previously identified a Plasmodium falciparum (Pf) protein of unknown function encoded by a single-copy gene, PF3D7_1134300, as a target of antibodies in plasma of Tanzanian children in a whole-proteome differential screen. Here we characterize this protein as a blood-stage antigen that localizes to the surface membranes of both parasitized erythrocytes and merozoites, hence its designation as Pf erythrocyte membrane and merozoite antigen 1 (PfEMMA1). Mouse anti-PfEMMA1 antisera and affinity-purified human anti-PfEMMA1 antibodies inhibited growth of P. falciparum strains by up to 68% in growth inhibition assays. Following challenge with uniformly fatal Plasmodium berghei (Pb) ANKA, up to 40% of mice immunized with recombinant PbEMMA1 self-cured, and median survival of lethally infected mice was up to 2.6-fold longer than controls (21 vs. 8 d, P = 0.005). Furthermore, high levels of naturally acquired human anti-PfEMMA1 antibodies were associated with a 46% decrease in parasitemia over 2.5 yr of follow-up of Tanzanian children. Together, these findings suggest that antibodies to PfEMMA1 mediate protection against malaria.

Genome sequence, transcriptome, and annotation of rodent malaria parasite Plasmodium yoelii nigeriensis N67

BackgroundRodent malaria parasites are important models for studying host-malaria parasite interactions such as host immune response, mechanisms of parasite evasion of host killing, and vaccine development. One of the rodent malaria parasites is Plasmodium yoelii, and multiple P. yoelii strains or subspecies that cause different disease phenotypes have been widely employed in various studies. The genomes and transcriptomes of several P. yoelii strains have been analyzed and annotated, including the lethal strains of P. y. yoelii YM (or 17XL) and non-lethal strains of P. y. yoelii 17XNL/17X. Genomic DNA sequences and cDNA reads from another subspecies P. y. nigeriensis N67 have been reported for studies of genetic polymorphisms and parasite response to drugs, but its genome has not been assembled and annotated.ResultsWe performed genome sequencing of the N67 parasite using the PacBio long-read sequencing technology, de novo assembled its genome and transcriptome, and predicted 5383 genes with high overall annotation quality. Comparison of the annotated genome of the N67 parasite with those of YM and 17X parasites revealed a set of genes with N67-specific orthology, expansion of gene families, particularly the homologs of the Plasmodium chabaudi erythrocyte membrane antigen, large numbers of SNPs and indels, and proteins predicted to interact with host immune responses based on their functional domains.ConclusionsThe genomes of N67 and 17X parasites are highly diverse, having approximately one polymorphic site per 50 base pairs of DNA. The annotated N67 genome and transcriptome provide searchable databases for fast retrieval of genes and proteins, which will greatly facilitate our efforts in studying the parasite biology and gene function and in developing effective control measures against malaria.

Plasmodium vinckei genomes provide insights into the pan-genome and evolution of rodent malaria parasites

BackgroundRodent malaria parasites (RMPs) serve as tractable tools to study malaria parasite biology and host-parasite-vector interactions. Among the four RMPs originally collected from wild thicket rats in sub-Saharan Central Africa and adapted to laboratory mice, Plasmodium vinckei is the most geographically widespread with isolates collected from five separate locations. However, there is a lack of extensive phenotype and genotype data associated with this species, thus hindering its use in experimental studies.ResultsWe have generated a comprehensive genetic resource for P. vinckei comprising of five reference-quality genomes, one for each of its subspecies, blood-stage RNA sequencing data for five P. vinckei isolates, and genotypes and growth phenotypes for ten isolates. Additionally, we sequenced seven isolates of the RMP species Plasmodium chabaudi and Plasmodium yoelii, thus extending genotypic information for four additional subspecies enabling a re-evaluation of the genotypic diversity and evolutionary history of RMPs.The five subspecies of P. vinckei have diverged widely from their common ancestor and have undergone large-scale genome rearrangements. Comparing P. vinckei genotypes reveals region-specific selection pressures particularly on genes involved in mosquito transmission. Using phylogenetic analyses, we show that RMP multigene families have evolved differently across the vinckei and berghei groups of RMPs and that family-specific expansions in P. chabaudi and P. vinckei occurred in the common vinckei group ancestor prior to speciation. The erythrocyte membrane antigen 1 and fam-c families in particular show considerable expansions among the lowland forest-dwelling P. vinckei parasites. The subspecies from the highland forests of Katanga, P. v. vinckei, has a uniquely smaller genome, a reduced multigene family repertoire and is also amenable to transfection making it an ideal parasite for reverse genetics. We also show that P. vinckei parasites are amenable to genetic crosses.ConclusionsPlasmodium vinckei isolates display a large degree of phenotypic and genotypic diversity and could serve as a resource to study parasite virulence and immunogenicity. Inclusion of P. vinckei genomes provide new insights into the evolution of RMPs and their multigene families. Amenability to genetic crossing and transfection make them also suitable for classical and functional genetics to study Plasmodium biology.

Erythrocyte membrane antigen frequencies in patients with Type II congenital smell loss

ObjectiveThe objective of this study was to determine whether there are genetic factors associated with Type II congenital smell loss. Study designThe expression frequencies of 16 erythrocyte antigens among patients with Type II congenital smell loss were determined and compared to those of a large control group. MethodsBlood samples were obtained from 99 patients with Type II congenital smell loss. Presence of the erythrocyte surface antigens A, B, M, N, S, s, Fya, Fyb, D, C, c, E, e, K, Jka, and Jkb was analyzed by blood group serology. Comparisons of expression frequencies of these antigens were made between the patients and a large control group. ResultsPatients tested for the Duffy b antigen (Fyb haplotype) exhibited a statistically significant 11% decrease in expression frequency compared to the controls. There were no significant differences between patients and controls in the expression frequencies for all other erythrocyte antigens (A, B, M, N, S, s, Fya, D, C, c, E, e, K, Jka, or Jkb). ConclusionsThese findings describe the presence of a previously unrevealed genetic tendency among patients with Type II congenital smell loss related to erythrocyte surface antigen expression. The deviation in expression rate of Duffy b suggests a target gene and chromosome region in which future research into this form of congenital smell loss may reveal a more specific genetic basis for Type II congenital smell loss.

Autoimmunity, phospholipid-reacting antibodies and malaria immunity.

Several questions regarding the production and functioning of autoantibodies (AAb) during malaria infection remain open. Here we provide an overview of studies conducted in our laboratory that shed some light on the questions of whether antiphospholipid antibodies (aPL) and other AAb associated with autoimmune diseases (AID) can recognize Plasmodia antigens and exert anti-parasite activity; and whether anti-parasite phospholipid antibodies, produced in response to malaria, can inhibit phospholipid-induced inflammatory responses and protect against the pathogenesis of severe malaria. Our work showed that sera from patients with AID containing AAb against dsDNA, ssDNA, nuclear antigens (ANA), actin, cardiolipin (aCL) and erythrocyte membrane antigens recognize plasmodial antigens and can, similarly to monoclonal AAb of several specificities including phospholipid, inhibit the growth of P. falciparum in vitro. However, we did not detect a relationship between the presence of anti-glycosylphosphatidylinositol (GPI) antibodies in the serum and asymptomatic malaria infection, although we did register a relationship between these antibodies and parasitemia levels in infected individuals. Taken together, these results indicate that autoimmune responses mediated by AAb of different specificities, including phospholipid, may have anti-plasmodial activity and protect against malaria, although it is not clear whether anti-parasite phospholipid antibodies can mediate the same effect. The potential effect of anti-parasite phospholipid antibodies in malarious patients that are prone to the development of systemic lupus erythematosus or antiphospholipid syndrome, as well as the (possibly protective?) role of the (pathogenic) aPL on the malaria symptomatology and severity in these individuals, remain open questions.

Occurrence of tick-transmitted pathogens in dogs in Jos, Plateau State, Nigeria

BackgroundCanine babesiosis caused by Babesia rossi, transmitted by Haemaphysalis elliptica in South Africa, has also been reported from Nigeria. Although H. leachi (sensu lato) is widespread in sub-Saharan Africa, published literature on the occurrence of canine babesiosis is meagre. It has been postulated that the genotype of Babesia rossi Erythrocyte Membrane Antigen 1 (BrEMA1) may be linked to virulence of the specific isolate. The primary objective of this study was to detect and characterise tick-borne pathogens in dogs presented to a veterinary hospital using molecular techniques. In B. rossi-positive specimens, we aimed to determine whether the BrEMA1 gene occurred and to compare genotypes with those found in other isolates. Lastly, we wished to identify the tick species that were recovered from the sampled dogs.MethodsBlood specimens (n = 100) were collected during January to March 2010 from domestic dogs presented at an animal hospital in Jos, Plateau State, Nigeria. They were screened for the presence of Babesia/Theileria and Ehrlichia/Anaplasma genomic DNA using PCR and Reverse Line Blot (RLB) assays. Positive B. rossi specimens were tested for the presence of the BrEMA1gene using an RT-PCR. In addition, ticks were collected from dogs found to be infested during sampling.ResultsOn RLB, 72 (72%) of the specimens were positive for one or more haemoparasites. Of the positive specimens, 38 (53%) were infected with B. rossi; 9 (13%) with Theileria sp. (sable); 5 (7%) with either Ehrlichia canis or Anaplasma sp. Omatjenne, respectively; 3 (4%) with Theileria equi; and 1 (1%) with B. vogeli and E. ruminantium, respectively. Co-infections were detected in 13 (18%) of the specimens. Results of RT-PCR screening for the BrEMA1 gene were negative. A total of 146 ticks belonging to 8 species were collected and identified: Rhipicephalus sanguineus 107 (73%), Haemaphysalis leachi (sensu stricto) 27 (18%), R. turanicus 3 (2%), and Amblyomma variegatum, H. elliptica, R. lunulatus, R. muhsamae and R. senegalensis 1 (1%), respectively.ConclusionsUp to 8 tick-borne pathogens possibly occur in the dog population at Jos, with B. rossi being the most prevalent. The absence of the BrEMA1 gene suggests that B. rossi occurring in that area may be less virulent than South African isolates.

Orthotopic Liver Transplantation After Successful Treatment With Anti-CD20 Monoclonal Antibody (Rituximab) for Severe Steroid-Resistant Autoimmune Hemolytic Anemia: A Case Report

Chronic hepatitis C virus (HCV) infection has been associated with a wide number of immunologic disorders, ranging from clinically silent laboratory abnormalities (eg, autoantibody positivity) to severe systemic diseases (eg, cryoglobulinemic vasculitis). Autoimmune hemolytic anemia (AIHA), due to the production of antibodies against erythrocyte membrane antigens, is an uncommon extrahepatic manifestation in the setting of chronic hepatitis C. Herein we have reported the case of a 57-year-old woman with decompensated HCV-related cirrhosis awaiting orthotopic liver transplantation (OLT) who experienced severe AIHA. After 1 month of treatment with prednisone (1 mg/kg body weight/d), there was no significant amelioration of anemia. Rituximab, an anti-CD20 monoclonal antibody that depletes B-lymphocytes reducing serum immunoglobulins, was initiated (375 mg/m 2 IV, weekly for 4 weeks) with a prompt, sustained increase in hemoglobin. The drug was well tolerated; it did not interfere with the course of the liver disease. Thirty-one months after rituximab therapy with resolution of AIHA, the patient successfully underwent OLT using immunosuppression with tacrolimus and low-dose steroids. The patient was discharged on postoperative day 36. No infectious event occurred in the postoperative period. At 18 months follow-up after OLT, there has been no infectious or hematological event. Our experience supported the safety of rituximab use in patients with advanced HCV-related liver disease before OLT.

Clinical course of autoimmune hemolytic anemia: an observational study

Autoimmune hemolytic anemia is characterized by the production of autoantibodies against erythrocyte membrane antigens. This study was carried out to identify the clinical, immunological and outcome characteristics of autoimmune hemolytic anemia patients treated at the (HC-UFMG) Pediatric Hematology Unit and the Hemocentro de Belo Horizonte. We evaluated 17 patients younger than 15 years old admitted from 1988 to 2003 were evaluated. Autoimmune hemolytic anemia diagnosis was based on the presence of acquired hemolysis and confirmed by positive direct Coombs polyspecific test results. Clinical, laboratory, and outcome data were obtained from patient records. The median age at diagnosis was 10.5 months. The direct Coombs polyspecific test was positive in 13 and negative in four patients. Monospecific testing was performed for 14 patients. The most frequent red cell autoantibody was IgG (five patients), followed by IgM in two. Thirteen patients had severe anemia and needed blood transfusions. Underlying diseases were identified in four patients: systemic lupus erythematosus, Hodgkin's lymphoma, autoimmune hepatitis and Langerhans cell histiocytosis. The remaining patients were classified as having primary disease. The median follow-up period was 11 months (5 to 23 months). Three children died, two after splenectomy and one with complications of the underlying disease. Autoimmune hemolytic anemia is rare in children and adolescents. Although patients usually respond to corticosteroids and/or immunoglobulin, fatal cases can occur. Prognosis is worse in patients with chronic underlying diseases.

Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]2GD3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]GT3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]2GD3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.

Biochemical characterization of canine blood group antigens: immunoprecipitation of DEA 1.2, 4 and 7 and identification of a dog erythrocyte membrane antigen homologous to human Rhesus

Despite the clinical significance of the canine blood group antigens, relatively little is known of the biochemistry of these molecules. In this study the canine blood group antigens DEA (dog erythrocyte antigen) 1.2, 4 and 7 were immunoprecipitated from red blood cells (RBC) bearing the corresponding blood group, and molecular weights of 85 kD (DEA 1.2), 32–40 kD (DEA 4) and 53–66 kD (DEA 7) assigned. DEA 1.2 and DEA 4 each appeared as a single band, whereas DEA 7 comprised three distinct bands (53, 58 and 66 kD). Polyclonal antisera specific for two peptides derived from the sequence of the human Rhesus blood group (Rh30A-C and Rh50A-C) were used in western blotting against carine and human erythrocyte membranes. The Rh30A-C antiserum identified a band of molecular weight 32 kD in both human and canine RBC, and the antiserum specific for Rh50A-C identified a band of 38–60 kD in human membranes and of 40–53 kD in canine RBC. This finding is consistent with conservation of areas of the Rhesus protein sequence between human and canine erythrocytes.

Proliferative responses of peripheral blood mononuclear cells from normal dogs and dogs with autoimmune haemolytic anaemia to red blood cell antigens

Autoimmune haemolytic anaemia (AIHA), one of the most common autoimmune diseases of the dog, is characterised by binding of autoantibody to erythrocyte membrane antigens leading to a decreased red blood cell (RBC) life-span. Failure of self-tolerance with activation of autoreactive T-lymphocytes is thought to play a key role in the initiation of such autoimmune events. Peripheral blood mononuclear cells (PBMC) were obtained from 11 clinically normal dogs, six clinically normal relatives of two littermate dogs which died from AIHA, and four dogs which had recovered from primary AIHA. Cells were stimulated in vitro with a panel of canine RBC-derived antigens (RBC membranes, glycophorin, spectrin, five 15-mer glycophorin peptides), the non-recall antigen keyhole limpet haemocyanin (KLH), and the mitogen concanavalin A (Con A). The kinetics of the proliferative responses to specific antigens were assessed by serially sampling the cultures from days 4 to 10. PBMC from all dogs responded strongly to Con A (day 2) and to KLH (maximal response on days 7 to 10) under appropriate culture conditions. Two of 11 normal dogs responded weakly to RBC membranes (mean stimulation index = 4.25). In contrast, PBMC from all dogs recovered from AIHA responded to RBC membranes (mean SI = 9.2 ± 2.5) and occasionally to other erythrocyte antigens. Similar responses were recorded with PBMC from dogs related to AIHA cases. It is considered that althouth normal individuals harbour erythrocyte-reactive lymphocytes, such cells are primed in dogs with AIHA or a genetic susceptibility to this disease.

Parasitized Erythrocyte Membrane Antigens of Plasmodium brasilianum: Relationships with the Ring-Infected Erythrocyte Surface Antigen of Plasmodium falciparum

An antigen, designated here as the parasitized erythrocyte membrane antigen (PEMA), is present in the erythrocyte membrane surrounding all intraerythrocytic stages of Plasmodium brasilianum. An antibody specific for PEMA appeared in 21 (50%) of 42 antisera from Saimiri sciureus monkeys naturally infected with P. brasilianum. Of these 42 sera, nine (21.4%) contained antibody to the ring-infected erythrocyte membrane antigen (RESA); of these nine sera, six did not react with PEMA. Sera of humans infected with P. malariae reacted with PEMA and RESA in a similar pattern; i.e., of 83 antisera, 71 (85.5%) reacted with PEMA and 30 (36%) reacted with RESA. Only one of these latter 30 sera were not reactive with PEMA. Of 167 sera from humans infected with P. falciparum but not P. malariae, 133 (79.6%) reacted with RESA; of these, 43 (25.7% of the total) reacted with PEMA but not RESA. Although PEMA was demonstrated with P. brasilianum and RESA with P. falciparum, neither PEMA or RESA could be demonstrated with P. malariae. Interactions of PEMA and RESA and the corresponding antibodies offer a method whereby the two morphologically similar quartan species, P. malariae and P. brasilianum, can be readily distinguished from each other and may furnish clues to genetic separation of the two and the mechanisms of interaction of quartan malaria and P. falciparum where they are coendemic.

Sequence diversity of the erythrocyte membrane antigen 1 in various strains of Plasmodium chabaudi

We have determined the complete genomic sequence of the Plasmodium chabaudi erythrocyte membrane antigen (PcEMA1) in 4 different parasite strains. The gene structure consisted of a short region encoding a signal sequence separated from the main coding region by an intervening sequence. The overall identity of the three P. chabaudi adami deduced protein sequences to their consensus was 100%, 99.8% and 88% for 556KA, DK and DS respectively, with a general pattern of increasing divergence from the N- to the C-terminus. The P. chabaudi chabaudi strain CB was 72% homologous to the P. chabaudi adami consensus sequence. A gene related to PcEMA1, designated PcEMA1-R, has been identified in the genome of P. chabaudi adami but not in P. chabaudi chabaudi. The partial sequence for this gene in P. chabaudi adami strain DS predicts that it could encode a truncated form of PcEMA1, but its status as a pseudogene or an independent, expressed gene has not been resolved.

Anti-erythrocyte membrane antibodies detected in sera of dogs naturally infected with Babesia gibsoni.

Due to the potential for anti-erythrocyte membrane antibodies as possible enhancers of erythrocyte destruction, the presence of serum anti-erythrocyte membrane antibodies in 31 dogs with Babesia gibsoni infection admitted to a veterinary hospital was investigated by an enzyme linked immunosorbent assay (ELISA) and immunoblotting analyses. This infection resulted in an increase of anti-erythrocyte membrane antibodies in 84% (IgG) and 74% (IgM) of 31 infected dogs, respectively. This was confirmed by the similarity in the protein profiles of the erythrocyte membrane antigens immunoblotted with rabbit antiserum to dog erythrocyte membrane antigens and infected dog serum. These results suggest the production of anti-erythrocyte membrane antibodies was induced by B. gibsoni infection.

Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites.

Plasmodium chabaudi: Polymorphic and nonpolymorphic epitopes of the antigen Pch105/RESA

The localization in the erythrocyte membrane of Pch105/RESA, the ring stage-infected erythrocyte surface antigen of Plasmodium chabaudi, the proposed analog to the vaccine candidate Pf155/RESA in P. falciparum, is here confirmed by the use of the immunogold technique in electron microscopy. Furthermore, a number of monoclonal antibodies to other P. chabaudi erythrocyte membrane antigens in the same molecular weight range as Pch105 were compared in different test systems. Data from immunoblotting of native and recombinant antigen as well as an inhibition ELISA indicate that Pch105 is identical to Pc96 and two other described antigens of 105 and 110 kDa. Pch105 could also be shown to have polymorphic epitopes, varying between different strains of P. chabaudi, without impact on the molecular weight.

Serological and biochemical characterization of monoclonal antibodies against red cell markers related to expression of lutheran blood group antigens

Monoclonal antibodies to erythrocyte membrane antigens have, prior to this investigation, led to identification of protein(s) which bear the Lub antigen [4] as well as to increased understanding of the effects of the in(Lu) gene [7, 8, 9, 10], a gene which acts in an autosomal dominant fashion to down-regulate expression of all Lutheran antigens as well as numerous other antigens recognized by human and murine monoclonal antisera [1, 2, 5]. This study was therefore designed to determine what, if any, further contribution could be made using the four submitted workshop antibodies toward the understanding of the biochemistry of Lutheran antigens and the mechanism of action of the In(Lu)

Biochemical Characterization of a Monoclonal Antibody to the H Type-2 Antigen: Comparison to Other ABH Antibodies

Monoclonal and polyclonal antibodies to the ABH blood group antigens were tested for their specificity to glycoproteins with ABH activity on immunoblots of solubilized erythrocyte membranes. Immunoblots were stained with monoclonal antibody G10 to the H type-2 carbohydrate structure or with commercially prepared monoclonal and polyclonal antibodies to A, B, and H blood group antigens. G10 antibody specifically stained antigens in the regions that contain the erythrocyte membrane bands 3 and 4.5; the staining was proportional to the expected H content of the erythrocytes (O greater than A2 greater than B greater than A2B greater than A1 greater than A1B). No specific staining was observed with membranes derived from Oh (Bombay) erythrocytes which lack the H type-2 structure. A commercially prepared monoclonal anti-H did not specifically stain erythrocyte membrane antigens. Monoclonal and polyclonal anti-A specifically stained bands from A and AB but not O, B, or Oh erythrocytes (A1 greater than A1B greater than A2 greater than A2B). Polyclonal anti-B serum specifically stained bands from B and AB but not O, A, or Oh erythrocytes (B greater than A2B greater than A1B). However, no specific staining was observed in tests with monoclonal anti-B. Monoclonal antibodies G10 and anti-A and polyclonal anti-A and -B blood typing sera will be useful in the further characterization of the molecular nature of the ABH antigens.

The pattern of reactivated fetal erythropoiesis in bone marrow disorders of childhood.

The expression of fetal characteristics of erythropoiesis (haemoglobin F concentration, haemoglobin A2 concentration, haemoglobin F cells, globin chain synthesis, carboanhydrase isoenzyme B, hexokinase isoenzymes, erythrocyte membrane antigens of the iI- and the ABH-system) was examined in red cells of twelve patients with different bone marrow disorders (juvenile chronic myeloid leukaemia (JCML), erythroleukaemia (EL), acute myelogenous leukaemia (AML), aplastic anaemia (AA) and Diamond-Blackfan anaemia (DB)). In JCML and EL all red cell parameters studied appeared to be fetal including the distribution of hexokinase isoenzymes. No fetal signs could be found in red cells of patients with AML. In two patients with DB who were treated by transfusion no fetal erythropoiesis could be detected. In one patient with DB under cortisone treatment i-antigen, ABH-antigens, haemoglobin F concentration, globin chain synthesis and hexokinase isoenzyme distribution were of the fetal or mixed type. In patients with AA only slight elevations of haemoglobin F were detectable. The nearly total reversion to fetal erythropoiesis in JCML and EL seems to be a part of the disorder itself, whereas in the other disorders the reactivation of fetal erythropoiesis could be the result of an erythropoietic stress.

Relationship between choline derivatives and mouse erythrocyte membrane antigens revealed by mouse monoclonal antibodies. I. Anticholine activity of anti-mouse erythrocyte monoclonal antibodies

Different clones of mouse hybridomas, derived from the fusion of unstimulated mouse peritoneal cells with mouse myeloma cells, producing IgM monoclonal antibodies directed against the membrane of bromelain-treated mouse erythrocytes (MRBC(Br)) have been previously established. We have recently shown that one of these hybridomas produce, in ascites, antibodies cross-reacting with phosphorylcholine derivatives (trimethylammonium (TMA) derivatives). In this work the cross-reactivity for TMA derivatives of the monoclonal antibodies produced by 4 anti-MRBC(Br) hybridomas have been studied at the cell level (plaque-forming cells). Phosphorylcholine, choline bromide and p-aminophenyl-trimethylammonium were found to be potent specific inhibitors of plaque formation (anti MRBC(Br)). The hemolytic activities of ascites and tissue culture supernatants were studied and their inhibition by TMA derivatives was determined. Immunoglobulins from ascites purified on TMA immunoadsorbant column were analyzed by two-dimensional gel electrophoresis, their spectrotype was compared to the spectrotype of immunoglobulins from tissue culture supernatants from the same hybridoma radioactively tagged by internal incorporation of [ 14C]leucine. It could be shown without ambiguity that the PTMA column retained an IgM with the same characteristics as the IgM secreted in vitro.