Biomedical subjects
Publications and source records attributed to D Olive.
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This report describes the molecular parameters of interaction of a new antibody (33B3.1) with the human membrane RIL2 expressed by ConA-activated T lymphocytes or allogeneic T-cell clones established from a rejected kidney allograft: the 33B3.1 immunoprecipitates a membrane protein of 55000 MW. It inhibits IL2-driven proliferation of activated T cells. This inhibition occurred in the nanomolar range when low concentrations of recombinant IL2 (rec-IL2) were used. The (125I)-33B3.1 binds in a specific way to a single class of receptor sites on activated T cells. The rate constants of association and dissociation at 37 degrees C of the labeled 33B3.1 were k*1 = 12 X 10(5) M-1 s-1 and k*-1 = 7 X 10(-4) s-1, respectively, and its equilibrium dissociation constant was KD = 0.65 nM. Maximal binding capacities were fairly variable among T-cell clones, as high as 300,000 sites/cell for some of them. Competition experiments demonstrate that the 33B3.1 and anti-Tac interact with the RIL2 in a competitive manner, suggesting that they recognize closely associated epitopes on the RIL2. However, the 33B3.1 inhibits the binding of (35S)-recombinant IL2 to its high affinity RIL2 in a noncompetitive way. The 33B3.1 seems therefore to interact with an epitope close but distinct from the IL2 binding site. Our data could suggest either that the 33B3.1 is able to convert high affinity RIL2 towards low affinity conformations or that there is more than one IL2 binding site per molecule of high affinity RIL2.
A clone of the interleukin 2-producing Jurkat leukemia cell line termed JA3 (surface phenotype, T3+, Ti+, T44+, T11+, T40+) has been used to induce and select cell variants lacking surface molecules involved in T-cell activation. Following 200 rad of gamma-radiation (1 rad = 0.01 Gy), cells were treated with monoclonal antibodies (mAbs) directed to T3, Ti, T44, or T11 antigen and complement. After growth of the residual cells in culture, "negative" cells were cloned under limiting conditions. Depending on the specificity of the mAb used for the immunoselection, three groups of variants were obtained. (i) The use of mAbs directed to T3 or Ti resulted in cell variants that expressed the T3 Ti- T44+ Leu1+ T11+ T40+ 4F2+ HLA class I+ surface phenotype. (ii) Immunoselection with anti-T44 mAb resulted in 2 variants that shared the T3- Ti- T44- Leu1- T11+ T40+ 4F2+ HLA class I+ phenotype. (iii) Cell treatment with anti-T11 mAb resulted in 15 variants characterized by the lack of T11 antigen expression and of all the other T-cell-specific surface antigens. Therefore, it appears that the different sets of JA3 cell variants, like T cells at discrete stages of intrathymic differentiation, may follow a coordinated expression of surface differentiation antigens. Analysis of the functional responsiveness of the three distinct groups of JA3 cell variants to different stimuli showed that all produced interleukin 2 in response to A23187 calcium ionophore plus phorbol 12-myristate 13-acetate. The first group of variants (T3- Ti-) did not respond to stimulation with anti-T3, anti-Ti, or anti-T44 mAbs. Eight of 9 did not respond to phytohemagglutinin either; however, all responded to appropriate stimulatory combinations of anti-T11 mAbs (and to calcium ionophore). The second group of variants (T3-, Ti-, T44-, T11+), similar to the first group, did not respond to anti-T3, anti-Ti, anti-T44 mAbs, and phytohemagglutinin, but they were fully responsive to anti-T11 mAb. The last group of variants (lacking all the T-cell-specific surface antigens) only responded to calcium ionophore A23187.
Pregnant female mice were randomly distributed in two groups and received twice a day by gavage either 25 mg/kg Dilantin in 0.2 ml distilled water or 0.2 ml distilled water till full term. The offspring were sacrificed on day 5. In the exposed group, the thymic cortex was reduced with a low mitotic activity in the lymphoid population while reticuloepithelial tissue was enlarged; numerous interdigitated reticular cells accumulate at the cortico/medulla border. In the spleen, the white pulp was enlarged because of lymphocytes accumulation in the periarteriolar sheets. In these two organs, the relative number of Thyl-2+, Lyt 1+ and Lyt 2+ T cell subsets were also modified showing especially a decrease in the thymus and an increase in the spleen of the Lyt 1+ cells. Since at the same time the thymus and the spleen of the females which reproduced the neonates are quite unchanged, it is concluded that the developing immune system of the fetal mouse is more susceptible to diphenylhydantoin than the immune system of the adult. This raises the question of the reversibility of this in utero acquired immune impairment.
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The effects of i.v. cytomegalovirus (CMV) immunoglobulin given for prophylaxis of CMV infections in recipients of allogeneic and autologous marrow transplants were evaluated in a randomized trial: 60 patients were randomly assigned to receive (30 patients) or not to receive (30 patients) CMV immunoglobulin for a period of 90 days after transplantation. As to the allografted patients, the cumulative incidence of asymptomatic and symptomatic CMV infections was significantly reduced in the CMV immunoglobulin-treated group as compared to the control group (56.5% versus 92.9%, P less than 0.05). No other statistically significant effect of CMV immunoglobulin could be found. In particular, the incidence of symptomatic CMV infections (including interstitial pneumonia), the mean delay of post-transplant viraemia and haematopoietic recovery were similar in the control and CMV immunoglobulin-treated groups. We conclude that prophylactic CMV immunoglobulin administration, as designed in our study, is no more than marginally effective and cannot be recommended without additional trials.
Longitudinal in vitro assays related to cell-mediated immunity were performed in patients following allogeneic (32) or autologous (15) bone marrow transplantation (BMT). In both groups of reconstituted patients, low CD4+/CD8+ T cell ratio and weak allogeneic mixed lymphocyte reactions were found in the first 6 months after BMT, progressively reaching values similar to controls (bone marrow donors or unrelated individuals). In contrast, a strong generation of allogeneic cytotoxic cells, assessed by the number of lytic units per 10(6) cells, was frequently found (18/38 patients tested in both groups) in the first 4 months, despite the quantitative deficit of the CD4+ subset. This in vitro differentiation was found to be independent of in vivo acute graft-versus-host disease (GVHD) and chronic GVHD in allo-transplanted patients. As also documented in autologous recipients, this observation suggests that this phenomenon could be, at least partially, related to the transplantation per se. Preliminary characterization of the effector cells indicates that they belong to the CD8+ subset and that their differentiation is interleukin-2-dependent. Experimental depletion of the CD4+ subset in normal subjects did not increase the number of lytic units in allogeneic cultures. This implies qualitative differences between BMT recipients and normal subjects, namely in CD8+ subset: i.e. that following BMT early CD8+ T cells appear to produce their own growth factor (IL-2), while in normal adult individuals, such autocrine CD8+ T cells, if present, are very rare.
In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MO1) recognizing an epitope of the MO1-alpha chain and the other (25.31) an epitope of the LFA1-alpha chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean +/- 1 SD) on plastic nuclon plates; this figure decreased to 33 +/- 5% and 23 +/- 6% of control adherence when the neutrophils had been pretreated with anti-LFA1-alpha (anti-alpha L) and anti-MO1-alpha (anti-alpha M), respectively. On another support (plastic culture chambers), 84 +/- 6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-alpha L or anti-alpha M was 10% and 43% of the control figure, respectively. These results show that adherence of neutrophils is dependent upon the plastic used. Moreover, inhibition of adhesion by the two MAbs was also dependent upon the support used for the assay, suggesting that MO1 and LFA1 may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-alpha L inhibited (congruent to 50%) locomotion more than did anti-alpha M (congruent to 25%), without altering chemoattractant-induced shape changes. These results suggest that the two MAbs inhibit chemokinesis but not chemotaxis. Many other adherence-associated functions, such as ingestion of opsonized Klebsiella pneumoniae, and cytotoxicity towards K/562 cells were decreased more by anti-alpha L than by anti-alpha M. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-alpha M than by anti-alpha L. Degranulation induced by zymosan or opsonized zymosan was altered by anti-alpha M only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-alpha M than by anti-alpha L, while degranulation induced by phorbol myristate acetate was not altered by either of the two Mabs.
Infection is the most important cause of mortality in leucopenic patients. A broad spectrum antibiotic therapy is imperative in febrile and neutropenic patients. In a multicentric study we have used ceftazidime (100 mg/kg/d) and netilmicin (6 mg/kg/d) in 88 children (fever greater than or equal to 38.5 degrees C, neutropenia less than 500/mm3) treated for acute leukemias (59), non Hodgkin lymphomas (13) or solid tumors (16). Median age was 7 years (2 months-16 years). In patients who continued to remain febrile, vancomycin (40 mg/kg/d) was added after 48 hours. The effective treatment was continued until a neutrophil count greater than 1,000/mm3. The first combination (ceftazidime + netilmicin) was effective in 64 children (73%) and the second combination (ceftazidime + netilmicin + vancomycin) in 11 patients. Bacteria were isolated in 39 children: Escherichia coli: 9, Staphylococcus epidermidis: 9, Staphylococcus aureus: 8, Streptococcus: 6, Pseudomonas aeruginosa: 3, Streptococcus pneumoniae: 1, Haemophilus: 1, Klebsiella pneumoniae: 1, Proteus: 1, Serratia: 1, Flavobacterium: 1. In these 39 patients, 30 became apyretic with ceftazidime and netilmicin and 6 after vancomycin. All blood culture were negative after the first combination. The median duration of antibiotic therapy was 14 days (5-9 days: 28, 10-20 days: 43, greater than 20 days: 17). There were no death, no superinfection. Tolerance was good without kidney or liver or biological perturbation. We conclude that the combination ceftazidime and netilmicin is effective in neutropenic children.
The authors report two cases of adenocarcinoma of the digestive system in children both of which were located in unusual sites. A child of 9 years old had an adenocarcinoma of the small bowel and a boy of 16 years who was known to have a long-standing hiatus hernia developed an adenocarcinoma of the oesophagus. In children these tumors have some characteristic features. They develop on a preexisting lesion. Evolution is rapid. From the pathological point of view, anaplasic and colloide patterns are frequent.
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Seven patients with immunodeficiencies (Wiskott-Aldrich syndrome, combined immunodeficiency, and osteopetrosis) were given a mouse monoclonal antibody against the alpha subunit of human leucocyte functional antigen (HLFA-1; CD18) to facilitate the engraftment of mismatched haploidentical related-donor bone marrow. Other conditioning included busulphan, cyclophosphamide, and antilymphocyte globulin. To prevent graft-versus-host disease the bone-marrow T cells were depleted with sheep erythrocyte rosetting and cyclosporin therapy was given. HLFA-1 antibody injections were well tolerated without side-effects except slight, transient fever (38-40 degrees) after the first injection. Engraftment was rapid in all seven patients. The regenerating leucocytes were of donor origin in all cases, and two patients have a mixed chimera. Two patients died from infections. The others are alive and well 60-395 days after transplantation. In a historical control group given the same treatment without anti-HLFA-1 infusion, only one of seven transplants partially engrafted; only two patients remain alive with autologous reconstitution but with uncorrected immunodeficiency.
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A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.
Functional studies, using mainly interleukin 2 (IL2)-dependent growth of human T cell lines or clones but also mixed lymphocyte cultures and mitogen T cell activation, allowed a collection of locally produced anti-IL2 receptor monoclonal antibodies (mAb) to be classified. They fell into two groups: one with strong to moderate inhibition of IL2, the other without any detectable functional activity in in vitro assays. Direct and sequential immunoprecipitation as well as peptide mapping confirm that all the mAb recognize the same surface molecule. The parameters responsible for such functional dichotomy were characterized: the main parameter was found to be linked to the epitopic cluster recognized on the molecule by the mAb. All functional mAb pertained to a given epitopic cluster and all the nonfunctional ones to an alternative cluster. Studies on mAb receptor and IL2 receptor interactions confirmed these findings and strongly suggest that functional mAb interact with a region on the IL2 receptor identical or very close to the site of ligand-receptor interaction. These data could facilitate the choice of mAb to be used in therapeutical approaches in vivo when ethical objections could be overcome by appropriate committees.
A large collection of monoclonal antibodies (mAb) directed against sheep red blood cell (SRBC) receptor (cluster of differentiation 2: CD2) were classified according to three criteria: their inhibitory effect on T cell-SRBC rosette formation; the epitopic cluster recognized on the CD2 molecule; their reactivity with resting or activated T cells. All mAb were then tested in a two by two checkerboard fashion for possible T cell mitogenicity, in presence or absence of a submitogenic dose of 12-O-tetra-decanoylphorbol 13-acetate (TPA), an agent known to be comitogenic for T cells, presumably in delivering a second signal, usually accessory cell dependent. The combined data demonstrate that in the absence of TPA only few pairs of mAb directed at distinct epitopes of the CD2 molecule were mitogenic for T cells (in approximately 30% of the population tested), and in the presence of a submitogenic dose of TPA the majority of T11.1 anti-CD2-mAb (9 out of 11) were strongly mitogenic for T cells of all individuals tested when paired with T11.2 anti-CD2 mAb. The two anti-T11.1 mAb, noncomplementary to anti-T11.2 mAb, were, however, strongly mitogenic when added to mAb of the T11.3 subgroup, represented by 1-Mono-2A6. Taken together, these data strongly suggest that the main characteristic of T cell mitogenesis triggered by anti-CD2 mAb is the requirement for a signal delivered simultaneously to two different epitopes of the CD2 molecule, whether these epitopes are T11.1, T11.2 or T11.3.