Analysis of 24-h recovery of transfused stored RBCs in recipient mice of distinct genetic backgrounds.

BACKGROUND AND OBJECTIVES: Human studies have demonstrated substantial donor-to-donor variation in refrigerated RBC storage with respect to several variables, including 24-h post-transfusion RBC recovery. However, the human studies leading to these observations are mostly performed using autologous transfusions of stored RBCs, thereby avoiding issues of infectious disease transmission and alloimmunization. Accordingly, one cannot distinguish whether variability in 24-h RBC recovery is due to alterations in RBC storage, differences in phagocytic activity of the recipient's reticuloendothelial system or both. Similar to humans, genetically distinct inbred mouse strains have substantial differences in RBC storage biology, including 24-h post-transfusion RBC recovery. MATERIALS AND METHODS: In this report, we juxtaposed 24-h recoveries in 15 distinct inbred strains of mice, holding the RBC donor constant to isolate transfusion recipient variation as an independent variable. Strains were chosen for differences in baseline reticulocyte count and haemoglobin, which may correlate to RBC life span and turnover. RESULTS: Unlike large differences observed in storage of RBCs obtained from different strains of mice, only subtle strain-to-strain differences were observed regarding 24-h post-transfusion RBC recoveries. CONCLUSIONS: These findings indicate that the murine strains examined are not likely to be useful in sorting out mechanisms of clearance of stored RBCs, and suggest that such mechanisms may be generally conserved in the strains of mice analysed.

Glucose-6-phosphate dehydrogenase deficiency in transfusion medicine: the unknown risks.

The hallmark of glucose-6-phosphate dehydrogenase (G6PD) deficiency is red blood cell (RBC) destruction in response to oxidative stress. Patients requiring RBC transfusions may simultaneously receive oxidative medications or have concurrent infections, both of which can induce haemolysis in G6PD-deficient RBCs. Although it is not routine practice to screen healthy blood donors for G6PD deficiency, case reports identified transfusion of G6PD-deficient RBCs as causing haemolysis and other adverse events. In addition, some patient populations may be more at risk for complications associated with transfusions of G6PD-deficient RBCs because they receive RBCs from donors who are more likely to have G6PD deficiency. This review discusses G6PD deficiency, its importance in transfusion medicine, changes in the RBC antioxidant system (of which G6PD is essential) during refrigerated storage and mechanisms of haemolysis. In addition, as yet unanswered questions that could be addressed by translational and clinical studies are identified and discussed.

Pancreatic islet cell surface glycoproteins containing Gal beta 1-4GlcNAc-R identified by a cytotoxic monoclonal autoantibody.

To investigate the autoimmune pathogenesis of spontaneously occurring diabetes mellitus in BB rats, spleen cells of newly diagnosed diabetic BB rats were fused with mouse myeloma cells. Hybridoma supernatants were screened for antibodies by indirect immunofluorescence and by 51Cr-release assays using the RINm5F rat insulinoma cell line. One clone, E5C2, produced an IgM kappa antibody that was cytotoxic for RINm5F cells, but not for other rat cell lines nor for primary rat islet cells. However, treatment of primary rat islet cells with neuraminidase exposed surface antigens and rendered the cells susceptible to complement-mediated lysis by antibody E5C2. Using immunostaining of glycolipids separated by thin-layer chromatography, hapten inhibition assays with defined carbohydrates, and Western blots, the antigens recognized by E5C2 on RINm5F cells were identified as glycoproteins with molecular weights of 60,000 and 68,000. The antibody recognizes a carbohydrate antigen containing the sequence Gal beta 1-4GlcNAc-R, which on RINm5F cells is predominantly hidden by covalently bound sialic acid. These studies raise the possibility that hidden antigenic determinants on islet cells exposed by a variety of means may be the target of autoimmune attack.

Use of mouse models to study the mechanisms and consequences of RBC clearance.

Mice provide tractable animal models for studying the pathophysiology of various human disorders. This review discusses the use of mouse models for understanding red-blood-cell (RBC) clearance. These models provide important insights into the pathophysiology of various clinically relevant entities, such as autoimmune haemolytic anaemia, haemolytic transfusion reactions, other complications of RBC transfusions and immunomodulation by Rh immune globulin therapy. Mouse models of both antibody- and non-antibody-mediated RBC clearance are reviewed. Approaches for exploring unanswered questions in transfusion medicine using these models are also discussed.

Inhibition of entry of HIV-1 in neural cell lines by antibodies against galactosyl ceramide.

Although the CD4 molecule is the principal cellular receptor for the human immunodeficiency virus (HIV), several CD4-negative cell lines are susceptible to infection with one or more HIV strains. These findings indicate that there are alternate modes of viral entry, perhaps involving one or more receptor molecules. Antibodies against galactosyl ceramide (galactocerebroside, or GalC) inhibited viral internalization and infection in two CD4-negative cell lines derived from the nervous system: U373-MG and SK-N-MC. Furthermore, recombinant HIV surface glycoprotein gp120 bound to GalC but not to other glycolipids. These results suggest a role for GalC or a highly related molecule in HIV entry into neural cells.

Glycolipid antigen expression in human lung cancer.

Several mouse monoclonal antibodies which recognize carbohydrate sequences distinguish between different types of human lung cancer immunohistologically. These antibodies bind to glycolipid antigens produced by the cancer cells. When these glycolipids are separated by thin-layer chromatography, immunostaining of the chromatograms yields complex patterns of antigen-positive bands. To determine whether glycolipid patterns are useful in the classification of lung cancer, 16 human lung cancer cell lines comprising the major histological types of primary lung cancer were studied. Neutral glycolipids and gangliosides were isolated and separated by thin-layer chromatography. Six anti-carbohydrate antibodies which recognize structurally related antigens were used for immunostaining. Neuraminidase treatment of the chromatograms was used to detect "cryptic" sialylated antigens. All the cell lines were unique with regard to the type, amount, and chromatography pattern of the glycolipid antigens produced. Small cell lung cancer cell lines synthesized the greatest variety of antigens, whereas cell lines with large cell cytology synthesized the least. Interestingly, there was an inverse relationship between expression of some glycolipid antigens and DNA amplification of the c-myc oncogene. This suggests that enhanced c-myc expression may influence the types of glycolipids expressed at the surface of lung tumor cells.

Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells.

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.

Restricted VH gene usage by murine hybridomas directed against the human N, but not M, blood group antigen.

The M and N human blood group antigens are complex glycopeptide determinants at the amino terminus of the red blood cell membrane glycoprotein, glycophorin A. The heavy and light chain variable region cDNA sequences were determined for seven murine monoclonal antibodies recognizing glycophorin A. Three of the antibodies were anti-M and four were anti-N. Each of the anti-M antibodies was composed of VH and VL regions derived from distinct germline gene families (VH1 (J558), VH4 (X24), VH5 (7183), VK5, VK8, and VK19). In contrast, all four anti-N heavy chains were composed of VH regions derived from the VH2 (Q52) germline gene family and all used the same J4 gene segment. In addition, two of the anti-N light chains were composed of VK regions from the VK8 germline gene family and used the J1 gene segment. Since each anti-N hybridoma was derived from different mice immunized by different protocols, these results suggest that the murine immune response to the N, but not the M, human blood group antigen is restricted.

Sialylated glycolipid antigens on human leukemic cell lines.

Many granulocyte-specific mouse monoclonal antibodies recognize the carbohydrate sequence 3-fucosyllactosamine, Ga 1 beta 1-4[Fuc alpha 1-3]GlcNAc, which occurs in cell-surface glycolipids and glycoproteins. In general, these antibodies bind to blast cells from most patients with acute myeloblastic leukemia, but not to those with acute lymphocytic leukemia. Neuraminidase treatment, however, increases exposure of this antigen on both myeloid and lymphoid cells. In the present study, the glycolipids from 13 lymphoid and nonlymphoid human cell lines were examined for the presence of unsialylated and sialylated 3-fucosyllactosamine sequences using a thin-layer chromatography immunostaining method. Nine of the cell lines were also tested by indirect immunofluorescence both before and after neuraminidase treatment. None of the six B-cell and T-cell lines had detectable neutral or sialylated glycolipid antigen. In contrast, six out of seven and five out of seven nonlymphoid cell lines had neutral and sialylated glycolipid antigens, respectively. These results agreed, in general, with those found by indirect immunofluorescence. They also represent the first direct demonstration of these sialylated glycolipids on human leukemic cells. Thus, in some cases increased antibody binding to neuraminidase-treated cells can be explained by the presence of sialylated glycolipid antigen.

CD59 signaling and membrane pores drive Syk-dependent erythrocyte necroptosis.

Mature erythrocytes (red blood cells (RBCs)) undergo the programmed cell death (PCD) pathway of necroptosis in response to bacterial pore-forming toxins (PFTs) that target human CD59 (hCD59) but not hCD59-independent PFTs. Here, we investigate the biochemical mechanism of RBC necroptosis with a focus on the mechanism of induction and the minimal requirements for such RBC death. Binding or crosslinking of the hCD59 receptor led to Syk-dependent induction of vesiculated morphology (echinocytes) that was associated with phosphorylation of Band 3 and was required for Fas ligand (FasL) release. FasL-dependent phosphorylation of receptor-interacting protein kinase 1 (RIP1) in combination with plasma membrane pore formation was required for execution of RBC necroptosis. RIP1 phosphorylation led to the phosphorylation of RIP3, which was also critical for RBC necroptosis. Notably, RBC necroptosis was mediated by FasL and not by other candidate inducers, including tumor necrosis factor alpha (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL). Other types of RBC damage, such as eryptotic damage, failed to induce necroptosis when combined with hCD59 crosslinking. This work sheds light on the requirements for this recently discovered PCD in RBCs and provides a clear picture of the biochemical mechanism of induction of RBC necroptosis.

Secretion of multiple forms of human luteinizing hormone by cultured fetal human pituitary cells.

Previous studies of the heterogeneity of human LH have employed LH stored within the pituitary gland. In this study we characterized LH secreted by dispersed fetal human pituitary cells. Chromatofocusing across a pH 9-6 gradient of the medium in which fetal pituitary cells had been grown yielded at least eight distinct peaks of LH immunoreactivity. The more basic LH peaks bound more strongly to Concanavalin-A-Sepharose than did the more acidic ones, suggesting that the more basic LH molecules contain more hybrid oligosaccharides, in which one antenna terminates in a mannose, and that the more acidic human LH molecules contain more complex oligosaccharides, in which both antennae terminate in negatively charged groups, sialic acid and/or N-acetylgalactosamine-sulfate. The biological/immunological activity (B/I) ratios of the secreted LH varied directly and dramatically with the pI, from 8.1 at pI 8.4 to 1.1 at pI 6.3. Secreted LH, therefore, exhibits similar heterogeneity of glycosylated forms as does stored LH, raising the possibility that the hormones that control overall LH secretion could affect the secretion of some isohormones more than others and thereby influence LH biological activity to a degree not predicted by measuring total LH immunoreactivity.

Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region.

We investigated Plasmodium falciparum parasitized erythrocyte binding to proteolytic fragments of thrombospondin and the effects of anti-thrombospondin monoclonal antibodies on this binding. Purified human platelet thrombospondin was cleaved by trypsin, chymotrypsin or thrombin. Fragments were separated by heparin-agarose affinity chromatography, removing the amino-terminal heparin-binding region. Trypsin at 5.0 micrograms ml-1 of thrombospondin cleaved thrombospondin to reduced 140 and 120 kDa fragments plus a reduced 25-kDa heparin-binding fragment. Infected erythrocytes bound to intact thrombospondin (3420 +/- 460 infected erythrocytes mm-2) and the carboxy-terminal fragment, yielding 120-140-kDa fragments on sulfhydryl reduction, but not to the 25-kDa fragment (144 +/- 104 infected erythrocytes mm-2 (mean +/- s.d., N = 4). Similar results were obtained with chymotrypsin and thrombin cleavage. When the anti-thrombospondin monoclonal antibody MA-I was added to immobilized thrombospondin prior to infected erythrocytes, adherence was inhibited by 99%. At the same concentration, MA-I inhibited adherence to C32 melanoma cells by only 35%. MA-I binds to a calcium-dependent structure at the C-terminal globular region of thrombospondin. Monoclonal antibody MA-II inhibited adherence to thrombospondin by 46%, while MA-III had no effect. These antibodies bind to the N-terminal globular region which includes the heparin-binding site and the segment connecting the two globular regions, respectively. The site(s) for infected erythrocyte binding on thrombospondin reside in the large, 140- or 120-kDa, proteolytic cleavage fragments, and not in the N-terminal heparin-binding region.

Endothelial cell antigens recognized by xenoreactive human natural antibodies.

Hyperacute rejection of vascularized, discordant xenografts is generally though to be initiated when natural antibodies of the recipient bind to endothelial cells of the donor organ. While rejection of such xenografts always occurs, the molecular targets of natural antibodies have not been elucidated. The aim of the experiments reported herein was to identify the molecules on porcine endothelial cells that would be recognized by human natural antibodies if a porcine organ were to be transplanted into a human (or rhesus). Toward the end, it was shown that the major components recognized by human serum on porcine endothelial cells are glycoproteins of 115kDa, 125kDa, and 135kDa (gp115/135). Reactivity with these glycoproteins was abrogated by enzymatic cleavage of N-linked oligosaccharides or of subterminal beta-D-gal residues suggesting that the determinants are located on oligosaccharides rather than on the polypeptide cores. The biological relevance of gp115/135 was suggested by experiments in which a similar series of components was shown to be recognized by rhesus natural antibodies and by the absorption of such antibodies by perfusion of porcine kidneys. The gp115/135 antigens were present on porcine platelets but not porcine RBC or lymphocytes. Nevertheless, purified RBC and lymphocytes absorbed human anti-gp115/135, suggesting that human natural antibodies recognize the same or crossreactive carbohydrate determinants expressed on the surface of a variety of cells.

Pancreatic islet-cell epitope recognized by an anti-sulphatide monoclonal antibody.

Insulin-dependent (Type 1) diabetes mellitus is recognized as an autoimmune disease and islet-cell antibody (ICA) is present in the majority of patients at diagnosis. ICA labels both beta and alpha cells and is believed to be directed against a glycolipid. In this study we examine the presence of sulphatide (3'-sulphogalactosylceramide) or closely related structures (sulpholactosylceramide and seminolipid) in islet cells by means of a monoclonal antibody, Sulph I. Histological examination of pancreatic tissue from Lewis and BB rats, and BALB/c and NOD mice showed a pronounced labelling of the islets of Langerhans with Sulph I. No staining of the exocrine pancreatic tissue, the heart, the liver, the adrenals, the thymus, the spleen or lymph nodes was seen, but staining of some tubular cells and glomerular cells in the kidney as well as of myelin in nerve cells was found. Cytological examination of isolated Lewis islet cells and their cell subpopulations, separated using a fluorescence-activated cell sorter (FACS), showed positive surface labelling of 97.3 +/- 2.2% (SD) of the beta cells and 84.4 +/- 3.0% of the non-beta cells. Thus, the epitope on the glycolipid sulphatide or closely related structures is--with the exception of neural and certain kidney tissue--specifically present in islet cells. Furthermore, the staining pattern of the antibody used, Sulph I, was equivalent to that of ICA.

Neutralization of Lewis blood group antibodies by synthetic immunoadsorbents.

The neutralization of antibodies to the Lewis blood group systems is important in confirming the presence of these antibodies and in investigating complex alloantibody problems. An improved method for neutralizing Lewis antibodies is described. Insoluble immunoadsorbents containing synthetic Lewis antigens are used to physically remove the antibody from serum. Thirty-five sera containing Lewis antibodies were neutralized completely using this technic; 23 non-Lewis alloantibodies were not inhibited. In contrast with current methods, this technic does not dilute the patient serum and results in an affinity purified serum sample.

DNA typing of the human MN and Ss blood group antigens in amniotic fluid and following massive transfusion.

Although red blood cell (RBC) antigen typing by agglutination is generally useful, several situations exist where this approach is difficult or impossible. For example, following a massive transfusion, a patient's residual RBCs are mixed with transfused normal donor RBCs. In this case, typing by hemagglutination primarily detects the antigens on the heterogeneous population of transfused RBCs. Agglutination testing is also of limited use for determining the phenotype of a fetus at risk for hemolytic disease of the newborn because fetal RBCs must be obtained by periumbilical blood sampling. Determining the genotype of an individual by analyzing genomic DNA isolated from peripheral blood nucleated cells or amniocytes is an alternative approach for determining the RBC antigen type. In this report, the allele specific polymerase chain reaction (AS-PCR) was used to identify the alleles at the MN and Ss loci that encode the corresponding antigens on glycophorin A (GPA) and glycophorin B (GPB), respectively. This method was used to type these alleles in peripheral blood samples obtained from normal individuals and from patients following massive transfusion. Of 23 peripheral blood specimens analyzed, all were correctly typed by this method. The allele specific polymerase chain reaction was also used to determine these alleles using amniotic fluid samples. Of 11 amniotic fluid specimens analyzed, 8 were correctly typed at both loci. Mistyping of three amniotic fluid specimens was explained by possible maternal blood contamination.

Thrombospondin binding by parasitized erythrocyte isolates in falciparum malaria.

Toward understanding the pathogenesis of vascular sequestration in falciparum malaria, we investigated binding of Plasmodium falciparum parasitized erythrocyte isolates to thrombospondin and other adhesive proteins. Blood samples with rings from 12 patients with falciparum malaria were cultured 30 hr until parasites were mature trophozoites and schizonts. All parasitized erythrocyte isolates bound to thrombospondin, but not to fibronectin, laminin, vitronectin, or factor VIII/von Willebrand factor. Parasitized erythrocyte binding varied among isolates, ranging from 192 to 6,725 per mm2, average 2,953. There was good correlation between trophozoite plus schizont % parasitemia and thrombospondin binding (r = 0.884, P less than 0.001). In two patients with stupor, 3,642 and 2,864 parasitized erythrocytes bound per mm2, in proportion to parasitemia, suggesting cerebral malaria is not due to increased binding affinity. These results indicate there is a conserved function among isolates from this geographic region, known to be antigenically diverse at the parasitized erythrocyte membrane surface. These results support the hypothesis that specific binding to an endothelial receptor, possibly involving thrombospondin, plays a role in vascular sequestration in falciparum malaria.

Studies of the receptors on melanoma cells for Plasmodium falciparum infected erythrocytes.

We investigated whether thrombospondin plays a role in the binding of Plasmodium falciparum parasitized erythrocytes to C32 melanoma cells. Twelve patient isolates bound variably to melanoma cells, with good correlation between the degree of binding to cells and binding to thrombospondin. With a synchronous preparation of asexual parasites, acquisition of the capacity to bind to thrombospondin occurred at the same parasite stage as binding to melanoma cells. Development of parasites to trophozoites and schizonts correlated with binding of parasitized erythrocytes to thrombospondin and melanoma cells. The infected erythrocyte receptor for thrombospondin was destroyed by mild trypsinization, as was the receptor for melanoma cells. Although these results suggest similarity in the melanoma cell receptor and thrombospondin receptor for infected cells, other results showed that thrombospondin cannot alone be the melanoma cell receptor. Binding to other melanoma cell lines did not correlate with thrombospondin secretion: the RPMI 8252 and G361 cell lines bound few or no infected cells, yet secreted 50-100% as much thrombospondin as C32 cells. Iodinated thrombospondin bound in similar amounts to C32 cells and to noncytoadherent C361 melanoma cells. Binding and nonbinding melanoma cells did not differ in quantity of surface thrombospondin by radioimmunoassay. Thus, although purified, immobilized, thrombospondin binds parasitized erythrocytes, expression of thrombospondin alone on melanoma cells is not sufficient to mediate adherence.

Analysis of the feline immune response to human myelin-associated glycoprotein.

Elucidation of the pathogenesis of demyelinating peripheral neuropathy associated with myelin-associated glycoprotein (MAG) binding IgM paraproteins requires an in vivo animal model of the syndrome. Multiple immunizations of cats with MAG in Freund's adjuvant did not produce an antibody response but four immunizations with MAG-iscom (Morein, B. et al. (1984) Nature, 308: 457-460) did induce IgM antibodies which bound to human MAG and cat peripheral nerve myelin. Despite the presence of antibody for a 13-month period, no neuropathy developed. At necropsy, the peripheral nerves were ultrastructurally normal and no antibody was detectable in the endoneurium. A competitive ELISA indicated that the cat and human IgM antibodies recognized different epitopes.

A novel, non-nested reverse-transcriptase polymerase chain reaction (RT-PCR) test for the detection of the t(15;17) translocation: a comparative study of RT-PCR cytogenetics, and fluorescence In situ hybridization.

BACKGROUND: The development of a rapid and simple reverse-transcription polymerase chain reaction (RT-PCR) assay is described that identifies the promyelocytic leukemia- retinoic acid receptor alpha (PML-RARa) hybrid messenger RNA (mRNA), a characteristic feature of acute promyelocytic leukemia (APL). METHODS AND RESULTS: Randomly primed complementary (cDNA) is synthesized from leukocyte RNA and amplified in the presence of Taq Gold in 2 separate reaction tubes containing primer pairs specific for intron 3 (bcr 3, long [L] form mRNA transcript) and intron 6 (bcr 1, short [S] form)/exon 6 (bcr 2, variant [V] form) breakpoints in PML, respectively. The different sized products generated from each RNA transcript (S, L, or V forms) are readily and unambiguously distinguishable after agarose gel electrophoresis without the need for either nested PCR or hybridization. The sensitivity of the assay is 1 in 10,000 to 1 in 100,000. The separate amplification of a b2-microglobulin transcript controls for adequate RNA and cDNA preparation. The newly developed assay was used clinically for the evaluation of 78 patients with APL. It was rapid and more sensitive than cytogenetic karyotyping, both for the diagnosis of APL and the assessment of minimal residual disease (MRD) after therapy. RT-PCR detected PML-RARa mRNA in all cases positive for the t(15;17) translocation by cytogenetics. However, as many as 50% and 80% of the diagnostic specimens and the specimens for MRD assessment, respectively, that were positive by RT-PCR were negative by cytogenetics. The ratio of cases with L-form to S-form PML-RARa fusion transcript was 2:1, whereas 3 cases (10%) had fusion sites in exon 6 of the PML gene (V forms). In addition, approximately 50% of the patients were diagnosed morphologically with microgranular M3V-type leukemia, but no significant correlation with PML breakpoints was found. CONCLUSION: The current assay is rapid, sensitive, and specific without using nested PCR or hybridization.