Chondroitin sulfate of thrombomodulin is an adhesion receptor for Plasmodium falciparum-infected erythrocytes.
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Animals↗
Biomedical subjects
L Edelman
Publications and source records attributed to L Edelman.
Obtaining a functional recombinant anti-rhesus (D) antibody using the baculovirus-insect cell expression system.
The cloning and production of a human anti-rhesus (Rh) D monoclonal antibody (mAb) using the baculovirus-insect cell expression system is described. This monoclonal recombinant antibody R.D7C2 derived from a human parental IgM lambda immunoglobulin was obtained after immortalization of lymphocytes by Epstein-Barr virus (EBV). The human heavy (VH) and light (VL) variable regions were cloned from the parental cell line and genetically fused to the human constant IgG1 heavy (H) and light (L) chain genes (gamma 1 and lambda, respectively). A recombinant baculovirus was constructed that directs the co-expression of genes encoding both genetically fused heavy and light chains under the control of two late and strong baculovirus promoters. After infecting the Spodoptera frugiperda (Sf9) insect cell line with this baculovector, a complete IgG1 mAb was secreted in the culture medium indicating that each immunoglobulin chain was correctly processed and assembled with a functional glycosylation into a tetrameric form. In vitro analysis showed that the functional properties of R.D7C2 using agglutination tests were efficient for the specific recognition of Rh-D-positive red blood cells (RBC). In addition, R.D7C2 showed effector functions of the gamma 1 heavy chain resulting in the lysis of Rh+ papain RBC by an antibody-directed cellular cytotoxicity mechanism. These results demonstrate that R.D7C2 can be produced in the baculovirus-insect cell expression system as a source for potential therapeutic application in the treatment of the haemolytic disease of the newborn.
Animals↗
Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death.
The severe outcome of the dengue (DEN) virus infection known as DEN hemorrhagic fever-DEN shock syndrome (DHF-DSS) is, in some cases, accompanied by liver injury. Councilman bodies observed in liver biopsies of DHF-DSS cases may correspond to hepatocytes in apoptosis. We show here that infection of the hepatoma cell line HepG2 with DEN type 1 virus induced cell death typical of apoptosis late in the virus cycle. The transcription factor NF-kappaB was activated concomitantly with viral protein synthesis and thus before the appearance of apoptotic cells. Inhibition of apoptosis was observed when DEN virus-infected cells were treated with NF-kappaB decoys, indicating the involvement of this transcription factor in induction of cell death. Thus, infected hepatocytes appear to be subject to apoptosis in vitro, and this may be a key element in the pathophysiology of hepatic failure associated with DHF-DSS.
Animals↗
Radioimagers as an alternative to film autoradiography for in situ quantitative analysis of 125I-ligand receptor binding and pharmacological studies.
Three radioimagers, the mu-imager, the beta-imager and the phosphorimager, were tested as alternatives to quantitative autoradiography on film, for receptor imaging and pharmacological in situ quantitative analysis. Two iodinated ligands 125I-interleukin-1 alpha and 125I-gonadotropin releasing hormone agonist were used for receptor characterization in mouse brain and pituitary sections. Due to the high number of the agonist receptors in rat pituitary gland, this tissue was used to compare measurements obtained from digital autoradiograms with classical gamma detector determination. This permits the evaluation of radioimager efficiency and absolute quantification. Radioimagers represent an improvement in terms of time of image acquisition. All the radioimagers are more sensitive than film for the detection of low levels of radioactivity. The spatial resolution provided by the mu-imager compares favourably with that obtained on film autoradiograms while digital autoradiograms from the phosphorimager and beta-imager did not show precise definition under our experimental conditions. Superimposition of histological structures from the stained sections with radiolabelled areas in the autoradiograms remains, at this time, the unique advantage of film. In conclusion, radioimagers represent an alternative to autoradiography on film or emulsion for in situ quantitative studies on tissue sections. They combine precise imaging for in situ binding studies with easy and direct access to counts in cpm. The improvement in radioimaging technology has, therefore, brought in situ analysis of iodinated ligand binding to the level of accuracy that is obtained with classical detectors of radioactivity.
Animals↗
Serum interleukin-10 in acquired immunodeficiency syndrome lymphoma patients. Seroco-Hemoco Study Group.
Interleukin-10 (IL-10) has multiple effects on lymphoid development, particularly as a stimulant of activated B-cell proliferation and differentiation. It is thought that IL-10 might play a role in the development of B lymphoid malignancies based on the observation that lymphomatous tissues from HIV+ patients contain numerous cells containing IL-10 mRNA as well as IL-10 protein. The aim of this study using an Elisa test was to analyze IL-10 in the serum of 18 HIV+ patients with non Hodgkin's B lymphoma (NHL) and compared the presence of this cytokine in the serum of 18 HIV+ patients without NHL. In this comparative study we also considered the different parameters such as the mode of contamination, sex, age and number of CD4 cells. 44% of the patients with HIV-related NHL had significant levels of IL-10 (> or = 12 pg/ml) in their serum, in comparison to the patients without NHL who did not show detectable serum IL-10.
Adult↗
Patterns of Epstein-Barr virus latent and replicative gene expression in Epstein-Barr virus B cell lymphoproliferative disorders after organ transplantation.
B cell lymphoproliferative disorders arising in organ transplant recipients (B cell posttransplant lymphoproliferative disorders [PTLD]) are generally associated with EBV. In previous reports, B cell PTLD were shown to express the full pattern of EBV latent genes, as in vitro-established lymphoblastoid cell lines. Although viral linear DNA was detected in 40% of lymphoproliferative disorders from immunocompromised hosts, immunophenotypic studies failed to detect late EBV replicative antigens. The aim of this study was to investigate the relationship of EBV latent gene expression in B cell PTLD to morphology, clonality, and immunophenotype, and to examine the replicative state of EBV in malignant cells. For this purpose, 9 cases of EBV-related B cell PTLD were analyzed. Immunoglobulin gene rearrangements were detected by Southern blot analysis. The presence of EBV was assessed by Southern blot and by in situ hybridization. B cell differentiation antigens, adhesion and activation molecules, and EBV latent and replicative gene expression were studied using immunohistochemistry techniques. We demonstrated that EBV-related B cell PTLD exhibited varying patterns of latent viral gene expression. Higher levels of adhesion molecules were detected in latent membrane protein 1 (LMP1) or LMP1 plus EBV nuclear antigen 2 (EBNA2)-positive tumors than in LMP1 and EBNA2-negative tumors. In contrast, there was no relationship between CD21 and CD23 expression and latent EBV phenotype. Activation of the EBV replicative cycle was highlighted by BamHI Z left frame 1 expression in 5 of 9 cases. Less frequent expression of late viral proteins suggested that the initiation of the EBV lytic cycle might not always lead to virions production.
Adult↗
Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin.
We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2-terminal heparin-binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (kappa), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 x 10(-8) M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25-30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50-60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin-Sepharose column at low salt concentrations. These fragments also reacted with MA-II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.
Animals↗
Epstein-Barr virus latent and replicative gene expression in post-transplant lymphoproliferative disorders and AIDS-related non-Hodgkin's lymphomas. French Study Group of Pathology for HIV-associated Tumors.
In acquired immunodeficiency, B-cell proliferation is usually associated with Epstein-Barr virus (EBV), implying the impairment of the normal control of EBV and EBV-infected cells. It has been assumed that EBV infection is latent in lymphoproliferative disorders. In order to determine the type of latency and to investigate whether any lymphoproliferative disorders enter into the lytic cycle, we analyzed the expression of latent and replicative EBV genes in 9 post-transplant lymphoproliferative disorders (PTLD) and in 23 EBV-positive AIDS-related non-Hodgkin's lymphomas (AR-NHL). The PTLD cases were categorized into polyclonal or monoclonal polymorphic tumors and monoclonal monomorphic tumors. The AR-NHL cases included large-cell/immunoblastic (LC/IB) and Burkitt's lymphoma (BL) groups. We demonstrated that varying patterns of latent-viral-gene expression are exhibited showing the 3 forms of latency. Polymorphic PTLD and LC/IB AR-NHL frequently expressed type II or III latency, whereas monomorphic tumors and BL AR-NHL showed type I latency. It is noteworthy that 3 cases of BL AR-NHL expressed latency II form. Induction of lytic cycle highlighted by the expression of BZLF1 occurred in 55.5% of PTLD, 36% of LC/IB and 22% of BL AR-NHL. In contrast, late viral proteins indicating productive cycle were present in 22% of PTLD, 14% of LC/IB, and were absent in BL cases. These data suggest that the impairment of EBV control permits disruption of latency, but the initiation of the lytic cycle may not always lead to viral production.
Gene Expression↗
[Corticosteroids in sepsis and in shock following sepsis].
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Adrenal Cortex Hormones↗
Quantitation of platelet glycoprotein IV (CD36) in healthy subjects and in patients with essential thrombocythemia using an immunocapture assay.
Glycoprotein IV (GPIIb, CD36) is a major platelet membrane glycoprotein which is thought to participate in a number of adhesive reactions and to mediate signal transduction. In order to measure the total content of GPIV in human platelets, we have developed a simple and sensitive solid-phase radioimmunoassay based on the immunocapture of GPIV from Triton X-100-solubilized platelets. FA6-152, a monoclonal antibody to GPIV was coated on microtiter plates and bound antigen was quantified with a radiolabeled polyclonal antibody to GPIV. Using purified GPIV as a standard, the coefficients of variation of the assay were found to be less than 10% at concentrations of GPIV ranging from 0.15 to 0.75 micrograms/ml. The assay was validated by the parallelism obtained between purified GPIV dose-response curves and those obtained with platelet lysates, indicating a similar antigenic activity for GPIV in both samples. The level of GPIV in platelets from healthy donors was 0.23 +/- 0.05 (mean +/- SD, n = 15) micrograms per 100 micrograms of platelet proteins and a mean value of 27,440 +/- 6,200 (SD) molecules per platelet was calculated. The radioimmunoassay could be used to discriminate between the high level of platelet GPIV in patients with essential thrombocythemia (mean +/- SD = 81,850 +/- 27,780 molecules/platelet; n = 8) and the normal GPIV level in patients with secondary thrombocytosis (mean +/- SD = 26,810 +/- 4,030 molecules/platelet; n = 5), thereby demonstrating the clinical usefulness of the assay. The specific increase in platelet GPIV in patients with essential thrombocythemia was confirmed by immunoblot analysis whereas no increase in platelet GPIb or GPIIb-IIIa was observed by this technique.
Adult↗
In vivo production of interleukin-10 by malignant cells in AIDS lymphomas.
Expression of the interleukin (IL)-10/BCRF1 gene was studied by in situ hybridization in tissue samples from acquired immunodeficiency syndrome (AIDS) lymphomas using a BCRF1 probe which also recognizes the human IL-10 sequence. Hybridization was detected in 8 out of 15 lymphomas. In contrast, the IL-10/BCRF1 gene expression was detected in only 1 out of 11 lymphomas from human immunodeficiency virus (HIV)-seronegative patients (p = 0.05). In AIDS lymphomas, the number of cells labeled with a BCRF1-specific probe was dramatically lower than that of cells labeled with the IL-10/BCRF1 probe. Thus, the IL-10 rather than the BCRF1 gene was expressed. Production of IL-10 was associated with that of IL-10 mRNA, as shown by immunodetection of the protein in numerous cells. In contrast, BCRF1-producing cells were rarely detected. Both in situ hybridization and immunochemical experiments indicated that malignant cells were involved in this IL-10 synthesis. IL-10 production in AIDS lymphomas was associated with the presence of Epstein-Barr virus (EBV) in lymphomatous cells (p = 0.02). As IL-10 is a potent growth factor for human B lymphocytes, these results suggest that IL-10 may stimulate the proliferation of malignant cells in an autocrine pathway in a number of AIDS lymphomas, and that EBV and HIV may synergistically trigger its production.
Gene Expression↗
Differential expression of two genes for 1-aminocyclopropane-1-carboxylate synthase in tomato fruits.
1-Aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) is the regulated enzyme in the biosynthetic pathway of the plant hormone ethylene. A full-length cDNA encoding this enzyme has been cloned from tomato fruits [Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. & Van Montagu, M. Proc. Natl. Acad. Sci. USA (1990) 87, 4859-4863]. We report here the complete nucleotide and derived amino acid sequences of a cDNA encoding a second isoform of ACC synthase from tomato fruits. The cDNAs coding for both isoforms contain highly conserved regions that are surrounded by regions of low homology, especially at the 5' and 3' ends. Gene-specific probes were constructed to examine the expression of transcripts encoding the two ACC synthase isoforms under two conditions of enhanced ethylene formation--namely, during fruit ripening and in response to mechanical stress (wounding). The level of mRNA encoding both isoforms, ACC synthase 1 and 2, increased during ripening. In contrast, wounding caused an increase in only the level of mRNA coding for ACC synthase 1. Blot analysis of genomic DNA digested with restriction enzymes confirmed that ACC synthase 1 and 2 are encoded by different genes.
Amino Acid Sequence↗
Platelet thrombospondin and glycoprotein IV abnormalities in patients with essential thrombocythemia: effect of alpha-interferon treatment.
Platelet aggregability and some biochemical parameters were evaluated in seven patients with essential thrombocythemia (ET) compared with seven patients with secondary thrombocytosis (ST). Defective platelet aggregation with one or more agonists was seen in five patients with ET whereas aggregation was increased in two other patients. In addition, three patients with ET demonstrated spontaneous platelet aggregation in citrated plasma. This was associated with increased level of thrombospondin (TSP) in the plasma membrane. Interestingly, the presence of a proteolyzed 160 kDa form of TSP was detected in all patients with ET, whereas it was never found in patients with ST. Furthermore, three patients with ET demonstrated increased levels of platelet surface glycoprotein IV (GP IV), the putative receptor for TSP in the plasma membrane. In two of these patients, this correlated with increased surface expression of TSP and spontaneous platelet aggregation. The results suggest a possible link between the increased number of plasma membrane GP IV molecules, the spontaneous expression of TSP on the platelet surface and platelet hyperaggregability in some ET patients. The levels of plasma membrane GP IV and platelet surface-associated TSP tended to be normalized during alpha-interferon treatment, whereas the presence of an altered form of TSP persisted. This last parameter might be of practical usefulness in the characterization of the disease, permitting a clear distinction from ST.
Aged↗
Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36.
The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.
Animals↗
Human monoclonal antibodies specific for the rabies virus glycoprotein and N protein.
Human monoclonal antibodies to rabies virus were established by Epstein-Barr virus infection of peripheral blood lymphocytes collected from a rabies-vaccinated donor, and fusion with a heteromyeloma line. Two human monoclonal antibodies, HUM1 and HUM2, both IgG2, reacted with the envelope glycoprotein of the rabies virus. The antibody HUM1 neutralized rabies virus (lyssavirus serotype 1) and Mokola virus (lyssavirus serotype 3), but did not neutralize European bat lyssavirus, suggesting that some common antigenicity exists between the glycoproteins of serotypes 1 and 3. In addition, this antibody neutralized a series of viruses resistant to neutralization by antibodies recognizing, in a murine system, antigenic sites I, II and III; however, it failed to neutralize viruses altered at site VI, indicating that human monoclonal antibody HUM1 is directed against antigenic site VI. The other human anti-glycoprotein antibody, HUM2, neutralized the European bat lyssavirus in addition to serotypes 1 and 3, but none of the resistant variant viruses altered at the sites mentioned above. A third human monoclonal antibody, HUM3 (IgM), was reactive with the internal nucleoprotein of the rabies virus. This antibody contained a murine light chain corresponding to the cytoplasmic murine chain not secreted in the heteromyeloma line. The potential use of monoclonal antibodies in post-exposure treatment of rabies is discussed.
Antibodies, Monoclonal↗
Immunopharmacological activity of monoclonal antidigitoxin.
A monoclonal antibody specific to digitoxin was developed and Fab fragments were prepared using the conventional papain method. The affinity constant was determined as 10(9)M-1 and the cross reactivity with digoxin was 2%. The Fab fragments were used for the reversal of acute digitoxin poisoning in rabbits (100% mortality). Fab fragments were administered over a 40 or 80 minute period just after digitoxin infusion. Rabbits treated with specific Fab fragments were protected by a bistoichiometrical dose. Evaluation of plasma digitoxin confirmed that the specific Fab fragments were able to extract and sequestrate digitoxin. The digitoxin-Fab fragment complexes were removed by renal excretion. Our data provide evidence of the benefit of monoclonal Fab fragments specific for digitoxin for the reversal of acute digitoxin poisoning.
Animals↗
Developmentally regulated expression of a 78 kDa erythroblast membrane glycoprotein immunologically related to the platelet thrombospondin receptor.
We have previously described a monoclonal antibody (FA6-152), obtained by immunizing mice with fetal human erythrocytes [Edelman, Vinci, Villeval, Vainchenker, Henri, Miglierina, Rouger, Reviron, Breton-Gorius, Sureau & Edelman (1986) Blood 67, 56-63]. The antibody labelled fetal, but not adult, erythrocytes and bound to both fetal and adult platelets and monocytes. In the present study we have characterized the antigen recognized by FA6-152 on human platelets and on cells of the erythroid lineage at different stages of maturation. FA6-152 precipitated a chymotrypsin-resistant 88 kDa sialoglycoprotein from both iodinated and periodate/NaB3H4-surface-labelled platelets which corresponds to glycoprotein IV, the platelet thrombospondin (TSP) receptor. After neuraminidase treatment, a shift of the apparent molecular mass from 88 kDa to 85 kDa was observed. Scatchard analysis revealed that 125I-FA6-152 bound saturably with high affinity to a single class of platelet binding sites (Kd 6.4 +/- 0.6 nM). The number of FA6-152 IgG molecules bound per platelet was 25,400 +/- 8,800 (n = 4) and did not change upon thrombin activation of platelets. At low doses of alpha-thrombin (0.025 unit), FA6-152 inhibited platelet aggregation as well as endogenous TSP binding to the platelet surface. Immunofluorescence labelling of bone-marrow cells and of cultures in vitro of burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) revealed that that FA6-152 antigen is a very early marker of erythroid differentiation and that its expression declines during maturation. Immunochemical identification of the FA6-152 antigen on fetal erythroblasts and fetal mature erythrocytes revealed a 78 kDa glycoprotein migrating just in front of the glycophorin A dimer. The antigen, which was absent from adult mature erythrocytes, was also detected in human erythroleukaemic (HEL) cells where FA6-152 precipitated two bands of molecular mass 85 and 88 kDa. Our data establish the existence of a previously unidentified 78 kDa erythroblast cell-surface glycoprotein whose expression is developmentally regulated during erythroid differentiation and which is immunologically related to the 88 kDa platelet TSP receptor.
Antibodies, Monoclonal↗
Induction and Accumulation of Heat Shock-Specific Poly(A) RNAs and Proteins in Soybean Seedlings during Arsenite and Cadmium Treatments.
Northern blot hybridization analyzes revealed that poly(A(+)) RNAs homologous to eight heat shock (HS)-specific cDNA clones were induced by arsenite (As) or Cd treatments. The mRNAs accumulated slower, and maximum accumulations were consistently lower than HS-induced levels. Prolonged treatment with low concentrations (50-100 micromolar) of As for 6 hours, or Cd for 12 hours, resulted in decreased accumulations of HS-specific mRNAs. This response resembled the ;autoregulation' observed during continuous 40 degrees C HS. However, no autoregulation was evident when soybean seedlings were exposed to high concentrations of As (250 micromolar) or Cd (1 millimolar) for 12 hours. The cDNA probe pCE54 detected a second higher molecular weight poly(A(+)) RNA following As or Cd treatments which accumulated concomitantly with the lower molecular weight HS-specific poly(A(+)) RNA. The patterns of low molecular weight HS polypeptides from in vitro translations induced by HS, As, and Cd, and analyzed by one-dimensional and two-dimensional SDS-PAGE, were similar but temporal differences were apparent. In addition to HS proteins, many control proteins were also detected in both in vitro and in vivo labeling patterns from As and, to a lesser extent, Cd treatments. The chemical agents used in this study apparently induced the accumulation and translation of HS messages in vivo but not in the selective manner as observed during HS treatment.
Journal Article↗