Characterization of a murine monoclonal antibody specific for the human P1 blood group antigen.

Four monoclonal antibodies (MAbs) directed against the P1 blood group antigen were produced by hybridomas obtained from mouse immunized with turtle-dove avomucoid. One of the MAb (154 IX B6) selected as a blood typing reagent agglutinated native P1 and Pk1 red cells with a high titer but was inactive against native P2, Pk2 and p erythrocytes. After papain treatment the reactivity towards P1 and Pk1 erythrocytes was enhanced whereas p erythrocytes remained unreactive. A weak cross-reactivity of the MAb with the Pk antigen was suspected since enzyme-treated Pk2 erythrocytes became significantly agglutinated. Further analysis of the antibody specificity was established by binding studies using neutral glycolipids prepared from P1 and P2 erythrocytes, affinity immunoabsorbents carrying known oligosaccharide structures and hapten inhibition with synthetic oligosaccharides. The MAb bound weakly to the Gal alpha 1-4Gal structure common to P1 and Pk antigens but had a marked preference for the P1 determinant (Gal alpha 1-4 Gal beta 1-4 GlcNAc) and the binding was abolished by prior treatment of oligosaccharide antigens by alpha(not beta)-galactosidase, which supports evidence that a terminal alpha-galactose residue is involved in the blood group P1 and Pk specificities. The MAb has a slightly broader specificity than the human anti-P1 counterpart but can be used safely for routine blood typing.

Soluble fibrinogen derivatives generated by thrombin: affinity for elastin.

When human citrated plasma is dialysed against a phosphate buffer containing Ca++, citrate anions are removed, thrombin is generated and soluble fibrinogen derivatives (fibrin monomers and/or soluble fibrin polymers) are formed. These derivatives are able to combine with human or bovine elastin to form a very stable addition product or adduct. The formation of the adduct is dependent on time, Ca++ and thrombin concentrations.

[Circulating hematopoietic cells. Results of 6 autologous grafts]. / Cellules hématopoïétiques circulantes. Résultats de 6 greffes autologues.

Six patients with acute non-lymphocytic leukemia underwent autologous transplantation of circulating stem cells collected during remission. They were given cyclophosphamide (120 mg/kg) and total body irradiation (1,000-1,200 rads) (five patients) or busulfan (16 mg/kg) and melphalan (140 mg/m2) before the transfusion of 6.7 X 10(8) nucleated cells/kg corresponding to 19.7 X 10(4) CFU-GM/kg. Granulopoietic engraftment was observed in every case and was influenced by the number of CFU-GM injected. Megakaryocytic recovery was obtained in four cases.

Characterization of an antibody to the integrin beta 3 subunit (GP IIIa) from a patient with neonatal thrombocytopenia and an inherited deficiency of GP IIb-IIIa complexes in platelets (Glanzmann's thrombasthenia).

Patient A.F. is a 28-year-old polytransfused woman with an inherited bleeding disorder, Glanzmann's thrombasthenia. An abnormal platelet function is linked to severe decreases in the platelet content of the integrins GP IIb and GP IIIa. In 1987 the patient gave birth to a child with severe anemia and thrombocytopenia. Serological tests revealed the presence of anti-platelet antibody together with an anti-Rhesus D. Western blotting identified a major antibody that reacted with a protein of 90-95 kDa present in platelets and endothelial cells. This was identified as the beta 3 integrin subunit (GP IIIa). Antibody-binding required intact disulfides, while controlled digestion with proteases showed the determinant(s) to be retained within chymotrypsin- (50, 63 kDa) and Staphylococcus aureus V8 protease-derived (25-38 kDa) fragments of GP IIIa. Direct binding assays performed in the presence of monoclonal antibodies specific for different epitopes on GP IIb-IIIa complexes confirmed that the epitope was exposed on intact platelets and revealed a specific inhibition of A.F. IgG binding by the monoclonal antibody, AP-3. Other tests confirmed that the antibody reacted independently of the PlA or Pen polymorphisms carried by GP IIIa. IgG purified from A.F. plasma by adsorption and elution from paraformaldehyde-fixed normal platelets or electrophoretically separated GP IIIa was an inhibitor of ADP-induced platelet aggregation. Unexpectedly, Western blotting showed trace amounts of abnormally migrating GP IIIa in A.F. platelets, which retained an ability to react with her antibody. This suggests that the patient has formed an autoantibody reactive with an active site of the beta 3 integrin subunit and linked to the development of neonatal thrombocytopenia.

Vibrational Structure of the (2)1Sigma+u-X(1)1Sigma+g Transition of the Ba2 Molecule.

Resonant two photon ionization (R2PI) technique was used to obtain the excitation spectrum of the Ba2 molecule. A group of 12 vibrational bands was found in the 740-764 nm region. As a result of mass selective detection, they were attributed unambiguously to the Ba2 molecule. By comparison to recent theoretical calculations, those bands were assigned to the (2)1Sigma+u-X(1)1Sigma+g transition; they may be fitted to give the following vibrational constants (in cm-1): omega"e = 33.2 +/- 0.2, omega"ex"e = 0.5 +/- 0.2, omegae = 65.2 +/- 0.2, and omega'ex'e = 0.4 +/- 0.2. Copyright 1998 Academic Press.

Polyreactivity of human monoclonal antibodies: human anti-Rh monoclonal antibodies of IgM isotype are frequently polyreactive.

The specific aim of this study was to characterize human anti-Rh monoclonal antibodies cross-reacting with self-antigens. We studied supernatants from man-mouse hybridomas and from lymphoblastoid cell lines. Man-mouse hybridomas were established by fusion of peripheral blood lymphocytes from healthy individuals recently immunized against Rh alloantigens, with mouse myeloma (or man-mouse heteromyeloma) cell lines. Lymphoblastoid cell lines were produced by Epstein-Barr virus induction of lymphocytes from identical sources. Of the 55 monoclonal alloantibodies studied, 11 also reacted with intracellular self-antigens as demonstrated by immunofluorescence assay on cryostat sections of human tissues. This cross-reactivity was mainly a property of monoclonal alloantibodies belonging to the IgM isotype (among the 11 cross-reacting mAbs 10 were IgM). The cross-reactivities of these monoclonal antibodies were ascertained by absorption of alloreacting antibodies with red blood cells. Similar results were obtained on a panel of purified cellular antigens by ELISA. The results confirm that during an immune response against a foreign antigen (alloantigen), B cells that produce polyreactive antibodies are not excluded from the pool of responding cells. Therefore, polyreactive autoantibodies present in sera from healthy individuals may be the result of an immune response against foreign antigens.

[Collection and freezing of blood stem cells in acute nonlymphoblastic leukemia]. / Collection et congélation des cellules souches sanguines au cours des leucémies aiguës non lymphoblastiques.

Blood-derived hemopoietic stem cells were collected using a continuous (10 patients) or a semi-continuous flow separator (2 patients) in some patients with acute non-lymphocytic leukemia. For ten out of those patients, five to seven leukaphereses were performed during a short period (median = 11 days) of marrow recovery following severe aplasia induced by an intensive chemotherapy. The mean number of CFU-GM cells collected per leukapheresis and per patient respectively was 6.7 X 10(4)/kg and 38.5 X 10(4)/kg. This latter number was similar to that obtained during a marrow harvest performed for a bone marrow transplantation, suggesting that high numbers of hemopoietic stem cells can be collected from the peripheral blood in leukemic patients and used for autologous transplantation.

[Production and characterization of monoclonal antibodies with P1 specificity]. / Obtention et caractérisation d'anticorps monoclonaux de spécificité anti-P1.

Four monoclonal antibodies, with P1 specificity were obtained after fusion of myeloma cells and spleen cells from mice immunized with turtle dove ovomucoid. Immediately after the fusion, the culture supernatants were studied for specificity with panels of erythrocytes and red blood cells sharing rare phenotypes (P1K, P2K, p) in the P system. After cloning, four monoclonal antibodies were produced, these antibodies strongly agglutinate P1 red blood cells, specially when they are used with 3% of dextran or with a 350 mmol/l concentration of sodium.