EP0599845A1 - Medicament contenant un agent inhibant in vivo l'apoptose pathologique et applications - Google Patents

Medicament contenant un agent inhibant in vivo l'apoptose pathologique et applications

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Publication number
EP0599845A1
EP0599845A1 EP92908880A EP92908880A EP0599845A1 EP 0599845 A1 EP0599845 A1 EP 0599845A1 EP 92908880 A EP92908880 A EP 92908880A EP 92908880 A EP92908880 A EP 92908880A EP 0599845 A1 EP0599845 A1 EP 0599845A1
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EP
European Patent Office
Prior art keywords
agent
cells
cell
lymphocytes
antibody
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EP92908880A
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German (de)
English (en)
French (fr)
Inventor
Jean-Claude Ameisen
Hervé GROUX
André Capron
Fabienne Ameisen
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
Original Assignee
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
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Publication of EP0599845A1 publication Critical patent/EP0599845A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/45—Non condensed piperidines, e.g. piperocaine having oxo groups directly attached to the heterocyclic ring, e.g. cycloheximide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2006—IL-1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex

Definitions

  • the present invention relates to the treatment of apoptosis of human cells characterized by the dysfunction or deficiency of certain cell populations.
  • It also relates to a method of selecting agents for the treatment of apoptosis in its pathological manifestations.
  • It also relates to methods for determining the capacity of cells of individuals affected by an HIV virus to respond to aggression by an infectious agent.
  • apoptosis is synonymous with the expressions "programmed cell death” or "activation-induced death”.
  • Apoptosis is a suicide mechanism of cells which has the particularity of being physiologically active. It is involved in the regeneration of tissues thus found in the involution during embryogenesis and adult life and the maturation of the embryonic immune system.
  • apoptosis differs from necrosis which is a response to attacks by the cell.
  • Apoptosis has pathological consequences that are all the greater since it can occur in cell populations that the organism is unable to regenerate in their adult form. differentiated, in particular memory lymphocyte cells and neurons.
  • the capacity for cellular regeneration of the organism can also be exceeded from a quantitative point of view.
  • pathological also relates to phenomena such as cell degeneration or aging involving an involution.
  • Apoptosis only concerns cells in the process of disappearing, and does not cause effects on the tissue and cellular environment: it is therefore not an apparent phenomenon, unless we really try to bring it into evidence.
  • TCR T cell receptor
  • lentiviruses causing pathological disorders resulting in an abnormal appearance of cellular suicides.
  • viruses SIV in monkeys By way of example of such lentiviruses, mention may be made of viruses SIV in monkeys, FIV in felines, BIV in cattle, VISNA in sheep, CAEV in goats and HIV in humans.
  • CD4 helper T cells CD4 helper T cells.
  • MHC-II major class II histo-compatibility complex
  • PWM mitogen pockeweed
  • anti-CD28 antibody increases the proliferation of T cells in patients infected with the HIV j virus, and thus allows a proliferative response of immature thymocytes to anti-CD3, and to two antibodies. anti-CD2.
  • HIV viruses vis-à-vis certain diseases or vis-à-vis certain antigens.
  • the applicants therefore set out to find an effective therapeutic agent, in particular with regard to diseases linked to the HIV virus.
  • the present invention therefore relates to a medicament containing an agent which in vivo inhibits pathological apoptosis.
  • an agent can be selected by a process comprising at least the steps:
  • a cell activating agent such as pokeweed mitogen (PWM)
  • PWM pokeweed mitogen
  • the retrovirus is an HIV.
  • HIV HIV
  • the virus in the presence of an agent according to the invention cannot teach cells apoptosis. This method, unlike the previous one, directly concerns the virus.
  • An agent according to the invention will be chosen from those capable of:
  • PHA phytohemagglutinin
  • Allogeneic irradiated cells are cells from an individual genetically different in terms of their HLA system.
  • Such a medicament is in particular intended for the treatment of diseases linked to retroviruses and in particular linked to lentiviruses, such as viruses.
  • Another subject of the present invention is the use of an agent as defined above for obtaining a medicament intended for the treatment of the previously mentioned diseases.
  • composition containing a pharmaceutically effective amount of an agent as defined previously in combination with one or more pharmaceutically acceptable diluents or adjuvants.
  • a pharmaceutical composition is intended for the treatment of diseases linked to retroviruses and in particular linked to lentiviruses.
  • the treatment of patients affected by these diseases will be done in a manner known per se and preferably by parenteral or intravenous route.
  • the present invention also relates to a method for determining the qualitative deficiency of the immunological repertoire of individuals vis-à-vis an infectious agent comprising at least the steps of:
  • the object of the present invention is also a method for determining the capacity of an individual's lymphocytes to respond to aggression by an infectious agent comprising at least the steps of:
  • the present invention further relates to a method of in vitro stimulation of lymphocytes from patients suffering from a disease linked to retrovirus by treatment of the lymphocytes isolated with an anti-CD28 antibody.
  • the present invention relates to a method for selecting an agent which in vivo inhibits pathological apoptosis comprising at least one step of bringing cells infected with a lentivirus such as an HIV virus into contact with an antigen, respectively in the presence and in the absence of said agent and consequently of stimulation by said antigen.
  • a difference in response between the cells cultured in the presence and in the absence of said agent would constitute an index of the inhibitory activity of this agent.
  • the anti-CD28 antibody preferably used for the implementation of the present invention is the mouse monoclonal antibody derived from the clone CLB-402 (catalog number JANSSEN 1989
  • FIG. 1a is a diagram illustrating the proliferation of cells of patients infected with the HIV 1 virus and of healthy individuals in the presence of phytohemagglutinin (PHA), of PWM, of staphylococcal enterotoxin B super-antigens (SEB) or of tetanus toxin (TT).
  • PHA phytohemagglutinin
  • SEB staphylococcal enterotoxin B super-antigens
  • TT tetanus toxin
  • FIG. 1b is a diagram showing the effect of the anti-CD28 antibody on the proliferation of cells of patients infected with the HIV 1 virus, in the presence of PWM, SEB or TT.
  • FIG. 2a illustrates the mortality of peripheral blood mononuclear cells (PBMC) of patients infected with the HIV ⁇ virus (columns 1) and healthy individuals (columns 2), in the presence of cycloheximide, cyclosporin A (CSA), anti-CD28 antibodies, PWM, or SEB.
  • PBMC peripheral blood mononuclear cells
  • FIG. 2b illustrates the cell mortality of PBMC cells of patients affected by the HIV 1 virus (columns 1) and of healthy individuals (columns 2) from which CD4 + T cells or CD8 + T cells have been eliminated, in the presence or in the absence anti-CD28, PWM or SEB antibodies.
  • FIG. 3 illustrates the results obtained after incorporation of tritiated thymidine in cells of patients affected by an HIV virus in the presence of PWM, or of PHA.
  • FIG. 4 is an autoradiogram representing the hybridization of total RNA of PBMC cells of patients affected by the HIV virus, cultured in the presence of PWM or of PHA, with a DNA probe complementary to the interleukin-2 receptor.
  • FIG. 5 is a snapshot of a DNA electrophoresis gel of PBMC cells from patients infected with the HIV 1 virus, cultured in the presence of PMW or SEB and of healthy individuals.
  • FIGS. 6a and 6b respectively represent photographs by electron microscopy of PBMC cells of healthy individuals and of patients affected by the HIV 1 virus.
  • Figures 7a and 7b are histograms corresponding respectively to the proliferation and cell death of cells cultured in a simple medium (column 1), in a medium supplemented with IL-2 (10 units / ml - column 2) and activated with bound CD5 antibody as a control (column 3) or by bound CD3 antibody (column 4 to 8) in the presence of recombinant IL-1 (50 units / ml) column 5, recombinant IL-2 (10 units / ml column 6), anti-IFNgamma antibody (serum dilution 1/800 - column 7) or cycloheximide (50 ⁇ / ml - column 8).
  • FIG. 7c is an electrophoresis gel of DNA extracted from medullary thymocytes cultured in a simple medium (column 1) or stimulated by CD3 (columns 4, 5 and 7) in the presence of recombinant Interleukin (column 5) or d anti-IFNgamma antibody (column 7).
  • FIG. 7d represents a dot blot analysis of RNA of medullary thymocytes stimulated by simple medium (column 1) with recombinant IL-1
  • FIG. 8 represents a gel for electrophoresis of DNA from T cells of chimpanzees infected with the HIV virus (columns 1 to 4) or of control chimpanzees
  • FIG. 9 represents a gel for electrophoresis in agarose of DNA of control macaque T cells stimulated with SEB (columns 1 and 2) of T cells of macaques infected with the SIV virus incubated in a simple medium (columns 3, 5 and 7) and T cells of macaques infected with the SIV virus, incubated with SEB (columns 4, 6 and 8).
  • FIG. 10 represents a gel for electrophoresis of DNA of T cells of macaques infected with the SIV virus incubated in simple medium (columns 1 and 3) or of medium + SEB columns 2 and 4), of T cells of green monkeys infected with SIV and incubated with SEB (columns 5 to 7) and green monkey T cells incubated with SEB (columns 8 and 9).
  • T lymphocytes from 38 patients infected with the HIV-1 virus and from 20 healthy individuals were tested in response to the polyclonal activators phytohemagglutinin, concanavalin A, mitogenic pockweed (PWM), anti-CD3 monoclonal antibody, and in response to the antigen stimulation of the tetanus toxin TT.
  • PWM mitogenic pockweed
  • TT-specific memory T cells are rare, and may have been eliminated in patients infected with the HIV virus, the response to the mobilization of T cell receptors by the use of enterotoxin B superantigens Staphylococcus (SEB) was also tested.
  • SEB enterotoxin B superantigens Staphylococcus
  • SEBs bind to MHC-II molecules and act with V ⁇ molecules of T cell receptors (TCR) expressed by up to 30% of normal T cells, and induce the proliferation of mature normal T cells (Marrack and Kappler, J Science 248, 705-711, 1990) and apoptosis in immature thymocytes (Genkinson et al. J. Immun. 19, 2175-20177, 1989).
  • TCR T cell receptors
  • HIV infection was attributed to 15 of the patients to homosexual relationships, to 14 of the patients to heterosexual relationships, to 7 of the patients to intravenous drug use and to 2 of the patients to blood transfusion.
  • the peripheral blood mononuclear cells were obtained on Ficoll-Hypaque and were cultured as described by Reed et al. (Proc. National Acad. Sci. USA 83, 3982-3986 (1986)).
  • the PBMCs are incubated at a cell density of 2.5 ⁇ 10 5 / ml for 3 days with culture medium without adjuvant, medium containing phytohemagglutinin (PHA) at 10 ⁇ g / ml, pokeweed mitogen (PWM) at 10 ⁇ / ml , staphylococcal enterotoxin B superantigen (SEB) at 1 ⁇ g / ml, or for 6 days with the antigen (TT) at 10 ⁇ g / ml.
  • PHA phytohemagglutinin
  • PWM pokeweed mitogen
  • SEB staphylococcal enterotoxin B superantigen
  • each circle represents the average of cultures carried out in triplica, the horizontal bars representing the average of the results for each treatment.
  • Trials have also been carried out by incorporating tritiated thymidine into T cells of 19 of the HIV infected patients (FIG. 1 b) in response to proliferation by PWM, SEB or TT, in the absence or in the presence of anti CD28 antibody.
  • the anti-CD-28 antibody is provided by the company JANSSEN (mouse monoclonal antibody derived from the clone CLB-402).
  • Cells were prepared and used as described for FIG. La and the anti-CD28 antibody was added at a concentration of 10 ⁇ g / ml.
  • FIG. 1 b shows that the anti-CD28 antibody increases the proliferation of control T cells in the presence of PWM or SEB.
  • the antibodies CD5 (A50, gamma 1, CD43 (B1B6, gamma 1), CD44 (P245, gamma 2a), and CD45R (ALB11, gamma 1, Immunotech) at concentrations of 10 ⁇ g / ml do not stimulate proliferation T cells from HIV + patients (results not shown in the figure).
  • FIG. 2 a illustrates the cell death of PBMC of patients infected with the HIV virus as well as controls, after in vitro culture in the presence of PWM and SEB.
  • results mentioned in FIG. 2a represent the average plus or minus the standard deviation of experiments carried out on 20 different patients infected with the HIV virus (columns 1) and 20 uninfected individuals (column 2). Cell mortality is demonstrated by the permeability to trypan blue.
  • PBMCs are cultured with an initial cell density of 2.5 10 5 / ml in
  • CsA cyclosporine A
  • FIG. 1 Figure 2b concerns similar experiments.
  • PBMC cells from an infected patient with the HIV virus (columns 1) and an uninfected individual (columns 2) were purified by elimination of CD4 + T cells (CD 8 cells) or CD8 + T cells (CD 4 cells) by incubation with respectively IOT4 or IOT8 antibody followed by incubation with magnetic particles coated with goat anti-mouse IgG antibody.
  • the quality of the elimination is measured by cytofluorometry (Epix Coulter Clone) and shows that the cell contamination is less than 1%.
  • the cells are cultured as described for FIG. 1 a).
  • the results represent the average of three measurements of cell mortality after 48 hours.
  • the proliferation is measured by incorporation of the tritiated thymidine after 3 days.
  • CD4 cells of uninfected individuals proliferate in response to PWM (5250 + 651 CPM) and SEB (21.642 + 2.317 CPM), while CD8 control cells and CD4 and CD8 cells from patients infected with HIV do not proliferate after 5 days.
  • anti-CD28 antibodies to the CD4 cells of patients infected with the HIV virus restores the capacity of these cells to proliferate during stimulation with PWM and SEB, but has no effect on the cells of patients. infected with unstimulated HIV or on control CD8 cells.
  • FIG. 3 illustrates the proliferation of PBMC cells of patients infected with the HIV virus and of healthy individuals after addition of PWM or of PHA, after incorporation of tritiated thymidine in the T cells of 3 patients infected with the HIV virus (ABC) and B ') and a healthy individual (D) in response to stimulation by PMW at 10 ⁇ g / ml (columns A, B and C, and column D) or by PHA at 10 ⁇ m / ml (column B ').
  • FIG. 4 illustrates the expression of the messenger RNAs coding for the interleukin 2 receptor (IL-2R) of PBMC of patients infected with the HIV virus and of healthy individuals after addition of PWM or of PHA.
  • the same patients (A, B, C, B 'and D) as in Figure 3 were repeated. This expression is measured on the same cells after 2 hours, 6 hours and 16 hours. These messenger RNAs were not detected before stimulation.
  • IL-2R interleukin 2 receptor
  • RNAzol Cina-Biotex, Friendwood, USA
  • the experiments were carried out by extraction of the total cellular RNAs from 10 7 PBMCs with RNAzol (Cinna-Biotex, Friendwood, USA) according to the manufacturer's instructions.
  • the total RNA samples (approximately 10 ⁇ g / ml) are fractionated according to their size in 1% agarose gels containing formaldehyde and are transferred to HYBOND-N filters (Amersham).
  • the filters are then hybridized with cDNA probes labeled with phosphate 32 (human IL-2R) and washed with a 0.1 SSC, 0.1% SDS solution at 55oC.
  • the relative sizes of the IL-2R mRNAs which hybridize with the probe are in agreement with the values of 3.5 kb previously published.
  • EXAMPLE 4 Degradation of cellular DNA.
  • FIG. 5 represents the ectrophoresis on DNA agarose gel extracted from PBMC of patients infected with the HIV virus or of healthy individuals after culture overnight in a culture medium with or without adjuvants.
  • EDTA 0.5% Triton X100
  • DNA from the supernatants is concentrated by precipitation at
  • Well 1 in Figure 5 corresponds to the molecular weight marker.
  • Well 2 corresponds to the DNA of PBMC cells from healthy individuals incubated with PWM.
  • Wells 3 to 11 correspond to the DNA of PBMC cells of 7 patients infected with the HIV virus incubated respectively with medium without adjuvant (well 3), medium comprising PMW (wells 4 to 6, 8 and 9), and medium comprising SEB (wells 10 and 11).
  • Wells 3 and 4 correspond to the same patient.
  • the figure shows that the DNAs from wells 4 to 6 and 8 to 11 have a degradation profile characteristic of apoptosis, the degraded DNA bands being multiples of 200 base pairs.
  • FIG. 6a represents a photograph by electron microscopy of PBMC of a patient infected with the HIV virus after an incubation of 24 hours in a medium without adjuvant.
  • FIG. 6b represents the same cells incubated in a medium containing 5 ⁇ g / ml of PWM.
  • influenza antigen is a commonly encountered antigen, it is possible that the three patients whose lymphocytes do not proliferate in the presence of anti-CD28 antibodies have already encountered it, and that the memory cells directed against this antigen have been eliminated thereby .
  • Lymphocytes were stimulated (OJ) after purification, by tetanus toxin (TT) by phytohemagglutin (PHA) and by the mitogen pockeweed (PWM).
  • OJ tetanus toxin
  • PHA phytohemagglutin
  • PWM mitogen pockeweed
  • the proliferation is measured by the incorporation of tritiated thymidine after 6 days (day 6).
  • the cells are cultured in the presence of tetanus toxin or the superantigen of staphylococcal B enteroxin (SEB) with or without anti-CD28 antibody.
  • SEB staphylococcal B enteroxin
  • the cells precultivated in the presence of the anti-CD28 antibody proliferate in the presence of the TT or SEB antigens.
  • Lymphocytes from patients infected with the HIV virus are stimulated either directly (JO) with phytohememaglutinin (PHA), the mitogen pockeweed
  • PWM staphylococcal enterotoxin B
  • SEB staphylococcal enterotoxin B
  • the cells are restimulated after 10 days under the same conditions as in the OJ.
  • lymphocytes The restoration of the capacity of lymphocytes to proliferate after a first stimulation with allogenic cells and in response to PWM and SEB was tested.
  • the monoclonal antibody CD3 does not induce the proliferation of thymocytes and leads after 48 hours to the death of approximately 50% of the thymocytes, as indicated in Table V below.
  • the cells were stained with a mixture of CD4-FITC antibody (LFL1 green fluorescence) and CD8-RD antibody (LFL2 red fluorescence) to perform a flow cytofluorometry analysis (Coulter Epies Profile II).
  • Cell proliferation after mobilization of TCRs with CD3 antibodies bound on a dish, in the presence or absence of IL-2 (10 units per ml) was determined by incorporation of tritiated thymidine ( 3 H-TdR). Thymidine was added in the last twelve hours of the third day.
  • the results represent the average of triplicate culture or a representative experiment among the three.
  • Cell death was determined 48 hours after stimulation by exclusion of trypan blue. The results and the percentages of cell death are chosen from three experiments.
  • Thymus were obtained from children less than two months after cardiac surgery.
  • the thymocytes were purified on a nylon sieve and were frozen until used.
  • the medullary thymocytes were isolated from a population of total thymocytes by negative selection with magnetic beads covered with a CD1 antibody (D47, IgG1 at l ⁇ .ml), according to the manufacturer's instructions (Biosys, France).
  • the CD4 + CD8- or CD8 + CD4- thymocytes were purified from medullary thymocytes by additional negative selection using respectively the CD8 monoclonal antibody (L533 IgG1 at 1 ⁇ / ml) or the CD4 antibody (0516, IgG1, at 1 ⁇ / ml).
  • Proliferation tests with fixed monoclonal antibodies (OKT3, IgG2a) or CD5 (A50, IgG2a) as controls were carried out. To determine cell death, the cells were harvested after 48 hours by pipetting and diluted in half with 0.1% trypan blue in PBS buffers. Living cells and dead cells were counted in a hemocytometer. The results are expressed as the percentage of dead cells.
  • the medullary thymocytes having been isolated by a negative selection, using the antibody CDla in all cases, and the antibodies CD46 and CD8 in the additional purifications, the three cell populations were contaminated by approximately 20% of immature thymocytes CD4- CD8- CD3-.
  • the cell mortality due to CD3 of total medullary thymocytes is associated with a DNA fragmentation characteristic of apoptosis as shown in Figure 7 C.
  • This figure is an agarose electrophoresis gel of DNA extracted from medullary thymocytes after different treatments.
  • the spinal cord thymocytes were cultured in a simple medium (column 1) or were stimulated by the bound CD3 antibody (columns 4, 5 and 7) in the presence of recombinant IL-1 (rIL-1) (TO U / ml, column 5), or in the presence of anti-IFN gamma (serum dilution 1/800, column 7).
  • This cell death is an active process which can be prevented by cycloheximine as shown in Figure 7b and cyclosporine A as shown in Table VI. l
  • apoptosis due to CD3 of medullary thymocytes is associated with the expression of the messenger RNA IFNgamma, with the secretion of IFNgamma, with the expression of the messenger RNA of the P55 chain of the IL-2 receptor, the absence of expression of the IL-2 gene and the absence of secretion of IL-2.
  • FIG. 7d represents the dot blot analysis of RNA of medullary thymocytes stimulated with a simple medium (column 1), a medium containing rILl ⁇ (50 units / ml, column 2), of the bound CD3 antibody (column 3) and CD3 antibody linked with rILl ⁇ (column 4).
  • the IL-2 autoradiogram was performed after 6 hours.
  • IFNgamma, IL-2 receptor and S-actin autoradiograms were performed after 6 p.m.
  • the probes were tested on a Northern Blot of RNA of thymocytes and each gives a specific signal without cross hybridization. The relative sizes of the messenger RNAs which hybridize to the probes are in agreement with the previously published values.
  • IL-2 prevents thymocyte death ( Figure 7b) and allows proliferation thymocytic in response to the CD3 antibody ( Figure 7a).
  • T-lymphocyte the addition of IL-1 to medullary thymocytes stimulated with CD3 allows the expression of messenger RNAs of IL-2 (Figure 7d) and the secretion of IL-2 (Table VI).
  • IL-1 does not affect the expression of IFNgamma messenger RNAs ( Figure 7d) or the secretion of IFNgamma (Table VI) in response to stimulation by CD3.
  • IFNgamma and IL-2 are added to thymocytes activated by CD3 and treated with CsA. Under these conditions, IFNgamma induces thymocyte death, while IL-2 induces thymocyte proliferation. When IFNgamma and IL-2 are added together, no cell death is observed and the thymocytes proliferate.
  • Table VII shows, for its part, that such a cell death program is not restricted to the stages of development of T cells, but is also present in the case of peripheral T cells, activated and mature humans.
  • a mature CD4 + T cell line specific for the tetanus toxin antigen behaves like human medullary thymocytes when stimulated by the CD3 antibody in the absence of other cells.
  • IFNgamma and / or IL-2 to peripheral mononuclear cells activated by CD3 and treated with CsA leads to the same results with the antigen-dependent T cell lines and the medullary thymocytes.
  • Table VIII below shows that neither the addition of IL-2 nor the addition of anti- Interferon gamma are not capable, unlike the antibody CD28, of preventing apoptosis of these lymphocytes, nor of restoring their proliferation in response to stimuli. IL-1 is also devoid of effect.
  • An antigen (tetanus, influenza) is presented to a T-CD4 + lymphocyte line by monocytes which have been preincubated with an HIV virus. The presentation of the antigen takes place in the presence of AZT to avoid infection of T-CD4 + lymphocytes. A normal lymphocyte proliferative response ensues.
  • the antigen when represented a second time to T-CD4 + lymphocytes by monocytes, even those not infected with the HIV virus, the lymphocytes die of apoptosis instead of proliferating.
  • FIG. 8 represents a gel for electrophoresis of DNA from T cells of chimpanzees infected with the HIV virus and incubated with SEB (columns 1 to 4) and of control chimpanzees, that is to say chimpanzees not infected with the HIV virus but whose T cells have been incubated with SEB (columns 5 and 6).
  • FIG. 9 corresponding to gels made with the DNA of T cells of control macaques stimulated with SEB (columns 1 and 2), of macaques infected with the SIV virus and whose cells were incubated in simple media (columns 3 , 5 and 7) and of macaques infected with the SIV virus and whose cells were incubated with SEB (columns 4, 6 and 8) shows that there is degradation of DNA and therefore apoptosis in the cells of macaques infected with the SIV virus when incubated with SEB.
  • Figure 10 shows that the African green monkey infected with the SIV virus does not develop apoptosis.
  • columns 1 to 4 correspond to the DNA of T cells of macaques infected with the SIV virus incubated with simple medium (columns 1 and 3) or with SEB (columns 2 and 4) while columns 5 to 7 correspond to T cells of African Green monkeys infected with SIV virus, incubated with SEB and columns 8 and 9 correspond to control African green monkey T cells, not infected with SIV, incubated with SEB.
  • This animal is therefore an interesting model for selecting therapeutic agents capable of preventing apoptosis and the onset of the disease.
  • This animal is all the more interesting since, unlike humans, where AIDS is declared in ten years, the macaque infected with the SIV virus has the particularity of developing AIDS in six months to a year and a half.
  • a protein synthesis inhibitor (cycloheximide) or cyclosporin A prevents apoptosis of T cells from patients infected with the HIV virus stimulated by PMW or SEB (FIG. 2a and FIG. 3).
  • the CD3 antibody induces cell proliferation and among the various co-signals (Interleukin 1, Interleukin 2, Interferon gamma, CD5 antibody, CD28, CD 43, CD44 and CD 45R or phorbol ester) which have been added to the T cells of patients infected with the HIV virus, only the CD28 antibody prevents death induced by PWM and SEB and restores proliferation to PMW, SEB and TT (FIG. 1b and Figure 2a).
  • the set of results seems to indicate that the CD4 T H cells of patients infected with the HIV virus are programmed in vivo for a suicide process by apoptosis after activation by defined stimulation, including the mobilization of T cell receptors (TCR) by the molecules presented by the major class II hiscompatibility complex.
  • TCR T cell receptors
  • tetanus-specific T cells appear to be present in patients infected with the HIV virus. Although such cells are too rare to allow their death to be demonstrated, the induction of apoptosis seems to be the most likely mechanism to explain their lack of response in vitro.

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EP92908880A 1991-03-27 1992-03-23 Medicament contenant un agent inhibant in vivo l'apoptose pathologique et applications Withdrawn EP0599845A1 (fr)

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FR9103739A FR2674433A1 (fr) 1991-03-27 1991-03-27 Medicament contenant un agent inhibant in vivo l'apoptose pathologique et applications.
FR9103739 1991-03-27
PCT/FR1992/000265 WO1992017193A1 (fr) 1991-03-27 1992-03-23 Medicament contenant un agent inhibant in vivo l'apoptose pathologique et applications

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GB9311132D0 (en) * 1993-05-28 1993-07-14 Eisai London Res Lab Ltd Control of cell death
FR2706772A1 (en) * 1993-06-22 1994-12-30 Vacsyn Sa Prevention and treatment of septic syndrome with an immunosuppressant, in particular cyclosporin.
US5935801A (en) * 1996-03-29 1999-08-10 Dana-Farber Cancer Institute Monoclonal antibody that detects apoptotic antigen
EP1057484A4 (en) * 1998-02-23 2002-11-20 Sagami Chem Res INHIBITORS OF CELL DEATH

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US4427684A (en) * 1982-09-24 1984-01-24 Ores Richard O Treatment and prevention of infection with herpes simplex and related viruses
US4814323A (en) * 1986-03-25 1989-03-21 Andrieu J M Process for the treatment and the prevention of AIDS and other disorders induced by the LAV/HTLV III virus
JPH0825890B2 (ja) * 1987-06-18 1996-03-13 呉羽化学工業株式会社 抗ウイルス剤
IL92382A (en) * 1988-11-23 1994-12-29 Univ Michigan Use of a ligand specific for CD28 in the manufacture of medicament

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