WO1998039413A1 - Models for haematopoiesis - Google Patents
Models for haematopoiesis Download PDFInfo
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- WO1998039413A1 WO1998039413A1 PCT/AU1998/000139 AU9800139W WO9839413A1 WO 1998039413 A1 WO1998039413 A1 WO 1998039413A1 AU 9800139 W AU9800139 W AU 9800139W WO 9839413 A1 WO9839413 A1 WO 9839413A1
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- csf
- factor
- cells
- haematopoietic
- deficient
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Definitions
- This invention relates to model systems for haematopoiesis .
- the invention relates to in vi tro and in vivo models which can be induced to produce neutrophils in the absence of G-CSF and/or GM-CSF.
- the model systems can be used to screen for putative haematopoietic, particularly neutrophil-promoting, activities. They may also be used to isolate, identify or characterise non-G-CSF, non-GM-CSF factors or cytokines, particularly neutrophilic stimulators.
- the biological regulators that govern the steady- state production of cells in the blood are believed, in part, to be the same factors that, in an emergency situation, serve to promote the production of large numbers of functionally active and terminally differentiated cells (Demetri et al 1991) .
- Two of the principal regulators that act on granulocyte-macrophage progenitor cells are granulocyte- acrophage colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF) .
- GM-CSF granulocyte- acrophage colony stimulating factor
- G-CSF granulocyte-colony stimulating factor
- CSFs The in vivo effects of CSFs have been studied in murine models by injecting pharmacological doses of CSF (Pojda et al 1990), by using transgenic mice (Lang et al 1987) and by studying mice with bone marrow cells which overproduce CSF (Marusic et al 1990) . While these studies have confirmed the activity of CSFs in vivo, they have not provided information about the physiological role of CSFs in steady- state or emergency haematopoiesis in animals, including man.
- mice deficient in either GM-CSF Stanley et al 1994
- G-CSF G-CSF
- the naturally-occurring op/op mutation provides mice that are deficient in macrophage-colony stimulating factor (M-CSF) , also known as colony stimulating factor-1 (CSF-1) , (Wiktor-Jedrzejczak, 1990) .
- M-CSF macrophage-colony stimulating factor
- CSF-1 colony stimulating factor-1
- mice deficient in one or more of these factors can be created by cross-breeding (International Patent Application No. W095/23862) .
- mice deficient in G-CSF still have morphologically recognisable neutrophils in the blood, but are strikingly neutropaenic .
- the invention provides an in vi tro model of haematopoiesis, comprising a co-culture of bone marrow-derived stromal cells and target haematopoietic cells, which co-culture increases haematopoiesis following exposure to an inactivated microorganism.
- the invention provides a model for emergency haematopoiesis.
- the co-culture may be a culture of stromal cells and bone marrow cells. These cells can be derived from a host having a disruption of a gene encoding a colony stimulating factor, such as G-CSF-deficient (-/-) mice or G-CSF/GM-CSF double deficient mice. Other types of mice which can be used include those described in W095/23862, for example mice simultaneously deficient in GM-CSF, G-CSF and CSF-1. Other animals or mice with gene disruptions may also be used, and the stromal cells and target haematopoietic cells may be derived from the same or different donor types.
- exposing irradiated stromal cells to haematopoietic cells in the presence of an inactivated microorganism results in the production of various combinations of cell types, depending on the nature and identity of the microorganism.
- the composition of the resulting cell population may depend on the combination of factors produced by the stromal cells in response to the inactivated microorganism.
- stromal cells from bone marrow of G-CSF (-/-) mice are co-cultured with bone marrow cells from mice of the same genotype.
- bone marrow cells from other animals of other genotypes may be used.
- the organism is preferably an infective microorganism, for example a microorganism such as a virus, a bacterium such as a mycobacterium, or a yeast.
- the infective organism can be any infectious agent that retains its antigenic properties following inactivation of its infectivity.
- any such microorganism may be used.
- the microorganism is a bacterium such as for example Mycobacterium tuberculosis, Mycobacterium bovis and the like, or a yeast such as yeasts of the genus Candida .
- the microorganism may be inactivated by any method which is capable of killing the microorganism, including but not limited to heat, chemical sterilants such as formaldehyde, glutaraldehyde or ethylenei ines, irradiation, ultrasonication, pressure disruption and the like.
- heat chemical sterilants
- strains of microorganisms which are temperature-sensitive may be used at the non-permissive temperature.
- the person skilled in the art will be aware of a wide variety of methods from which to choose the one which is most appropriate for a given micoorganism.
- the microorganism is Candida albicans .
- Candida albicans which has been killed by heat or chemicals, preferably by heat, is especially preferred.
- Haematopoiesis following exposure of the co- culture to killed Candida albicans is preferably characterised by neutrophilia, ie. by a significant increase in the number of neutrophils.
- the invention provides an in vivo model of haematopoiesis, comprising a mouse deficient in one or more haematopoietic factors, and which has been injected with a sub-lethal dose of a living microorganism.
- the microorganism is as described above.
- the factor has one or more of the following characteristics: a) ability to promote differentiation and multiplication of haematopoietic cells in the in vi tro model of the invention; b) the haematopoietic activity is not abrogated by antibody directed against known haematopoietic factors such as G-CSF, G-CSF, M-CSF (CSF-1) or S-CSF; c) has a molecular weight of greater than 10 kD; d) elutes as a peak on gel filtration using a
- the factor promotes differentiation of haematopoietic precursor cells into the granulocyte/macrophage and/or macrophage progenitor lineage. Most preferably the factor promotes differentiation into mature neutrophils. Most preferably the factor has the capacity to promote neutrophilia independent of the presence of G-CSF and/or GM-CSF.
- the factor of the invention is a protein or glycoprotein.
- Methods for purification of proteins and glycoproteins are extremely well-known in the art, and the person skilled in the art will be aware of a wide variety of suitable methods for purification of the factor to homogenity. Methods for identifying and isolating the gene encoding the factor are similarly well- known.
- methods for preparing antibodies, including monoclonal antibodies, directed against the factor of the invention are very widely known in the art, and methods of preparation of antibodies, antibody fragments such as Fab or F(ab) 2 fragments, and antibody analogues such as ScFv fragments using recombinant methods are routine in the art.
- the invention includes within its scope purified factors of the invention, nucleic acids encoding them, and antibodies, monoclonal antibodies, and ScFv constructs directed against them.
- Methods which may be used in the purification of factors, isolation of genes, and generation of antibodies include those set out in the U.S. provisional patent application by Ludwig Institute for Cancer Research, entitled “Dendritic Cell Factor” filed on 14 October 1997, the entire disclosure of which is incorporated herein by this reference.
- the invention provides a method of inducing an increase in neutrophil numbers in a mammal in need of such treatment, comprising the step of administering an effective amount of the factor of the invention to the mammal.
- a method of inducing neutrophil-promoting activity in a mammal in need of such treatment comprising the step of administering an effective amount of the factor of the invention to the mammal .
- the mammal may be a human, or may be a domestic mammal, including companion, farm and zoo mammals.
- the mammal may be at risk of infection, suffering from infection, or suffering from a condition which results in neutrophil deficiency. Any suitable route of administration may be used.
- the most suitable dosage and route of administration will depend on the route of the condition to be treated, and will be at the discretion of the attending physician or veterinarian .
- the activities of one or more cytokines or haematopoietic factors can be studied.
- the in vi tro and in vivo models of the invention can be used to identify neutrophilic factors.
- the invention provides a method of screening for factors or agents which are useful in the treatment of infection, comprising the step of testing a putative factor or agent for its ability to stimulate haematopoiesis in a model according to the invention.
- the activity of new factors can also be assessed, as well as combinations of such factors with known cytokines such as interleukins, known haematopoietic factors such as G-CSF or GM-CSF or the like.
- the in vi tro and in vivo effects of such combinations on haematopoiesis can also be investigated using the invention.
- This method of screening is particularly useful in identifying agents useful for treatment of immunosuppressed patients or in clinical settings where elevated numbers of neutrophils are desired
- the word “comprising” means “including but not limited to”, and that the word “comprises” has a corresponding meaning.
- the word “inactivated” in reference to a microorganism means that the microorganism is either killed or has its ability to infect a host abolished by a treatment such as heat or exposure to chemicals, but that the antigenic properties of the microorganism are retained.
- the word “inactivation” has a corresponding meaning.
- Figure 2 shows the total number of colony-forming cells, observed after 14 days in semisolid agar cultures, recovered from the non-adherent fraction of the 7 day bone marrow and stromal cell co-cultures from wild type and G-CSF deficient mice in the presence or absence of Candida albicans .
- Figure 3 shows the production of haematopoietic cells in Dexter cultures (Dexter et al , 1977) established using G-CSF-deficient stromal cells. Stromal cells were seeded with 2.5 x 10 6 G-CSF-deficient bone marrow cells together with 2.5 x 10 6 heat-inactivated Candida albicans ( Figure 3a) , or alone ( Figure 3b) . Haematopoietic cells are represented by bright cells in the foreground ( Figure
- Figure 4 shows levels of IL-6 in serum from G-CSF deficient mice and in wild-type control mice following infection by Candida albicans .
- Figure 5 shows the effect of various cytokines on the colony forming cells in bone marrows of G-CSF deficient mice ( Figure 5a) and in wild-type, control mice ( Figure 5b) .
- Bone marrow from each type of mice was isolated on days 0, 3 and 7 after Candida albicans infection and assayed for progenitor cells by soft agar assay. Colonies were scored 7 days after plating, and each value represents the mean of observations on 3 mice for each genotype .
- Figure 6 shows the results obtained when 10-fold concentrated conditioned medium from G-CSF deficient stroma stimulated with Candida albicans was fractionated on a Sephadex G-100 column.
- Figure 8 is another field from the same slide as that depicted in Figure 6, showing the presence of macrophages.
- the arrows indicate neutrophils.
- Figure 9 shows results obtained when metrizamide-fractionated bone marrow cells were incubated in the presence of factors and conditioned medium from G-CSF/GM-CSF double deficient stroma a) M-CSF (10 ng/ml) b) SCF (20 ng/ml) c) conditioned medium (1:4 dilution) d) conditioned medium (1:4 dilution ) + Ack-2 antibody e) conditioned medium (1:4 dilution) + anti-M-CSF antibody f) medium alone for 4 days at 37°C in a humidified atmosphere containing 5% C0 2 . The cells were stained with May-Grumwald Giemsa. The arrows indicate neutrophils.
- mice 8-10 week old G-CSF, G-CSF/GM-CSF double deficient and wild type sex-matched mice raised in microisolators were used for the study. Mice were bled by retro-orbital puncture 2 days before Candida challenge for baseline blood counts and for determining the levels of various cytokines. Mice were injected with 2.5 x 10 5 colony forming units (cfu) of Candida albicans through the lateral tail vein. Groups of mice were bled at various days after infection and thereafter sacrificed. Serum was separated immediately by centrifugation at 1000 x g for 10 min, aliquoted and frozen at -20°C for further use.
- cfu colony forming units
- Colony formation was stimulated by the following recombinant growth factors at the concentrations specified: human G-CSF (10 ng/ml) , murine GM-CSF (10 ng/ml) , murine IL-3 (10 ng/ml) , murine M-CSF (10 ng/ml), rat stem cell factor (SCF, 100 ng/ml).
- human G-CSF (10 ng/ml)
- murine GM-CSF (10 ng/ml)
- murine IL-3 10 ng/ml
- murine M-CSF 10 ng/ml
- SCF rat stem cell factor
- mice deficient in expression of G-CSF (G-CSF -/-), GM-CSF (GM-CSF -/-) or both G-CSF and GM-CSF (G-CSF/GM-CSF double deficient) were produced by targeted gene disruption and appropriate subsequent inbreeding or cross-breeding as described in W095/23682.
- Mice deficient in both G-CSF and IL-6 were produced by appropriate crosses with IL-6 -/- mice.
- Mice deficient in G-CSF and circulating Stem Cell Factor (SCF) are produced by appropriate crosses with Sl/Sl d mutant mice.
- SCF Stem Cell Factor
- Bioassays for IL-6, G-CSF, IL-3 , GM-CSF and SCF IL-6 assay IL-6 in serum or culture supernatant was measured by the 7TD1 proliferation assay as described in Brouckaert et al , 1989.
- G-CSF assay The G-CSF bioactivity was assayed by a cellular proliferation assay using Ba/F3 cells expressing a transfected human G-CSF receptor (designated Ba/f 19.1), which confers on Ba/F3 cells the ability to proliferate in the presence of both human and mouse G-CSF (Layton, Ludwig Institute for Cancer Research, Melbourne) . Briefly, test samples were serially diluted in 96-well flat bottomed plates and incubated for 48 h at 37°C in an atmosphere of 5% C0 2 in air with 10000 Ba/F19.1, in parallel with 10000 Ba/F3 cells. Dilutions of G-CSF were included as standards. After 48 h, the cells were pulsed with 0.5 ⁇ Ci of [ 3 H] -thymidine, harvested after 4h, and incorporation of [ 3 H] -thymidine assessed using a beta-scintillation counter.
- Example 1 Effect of Candida albicans on peripheral blood neutrophil counts and bone marrow neutrophil counts in G-CSF-deficient , G-CSF/GM-CSF double deficient and wild-type mice
- G-CSF/GM-CSF double deficient mice Cohorts of wild-type, G-CSF deficient and G-CSF/GM-CSF double deficient mice were infected with a sub-lethal dose (2.5 x 10 5 cfu) of live Candida albicans by injection into the lateral tail vein.
- Peripheral blood neutrophil counts were obtained 2 days prior to infection (termed day 0) and 4 and 7 days after challenge with the micro-organism. A striking degree of neutrophilia was evident in day 4 in the G-CSF deficient group, and this was maintained at day 7, as shown in Table la.
- Example 5 Bone Marrow Liquid Assay for Neutrophil- Promoting Activity
- Conditioned medium was generated from G-CSF- deficient stroma incubated with Candida albicans for 24 h. Initially this conditioned medium was tested for its colony stimulating activity (CSA) in a soft agar assay using bone marrow cells isolated from C57B1/6 mice. 25,000 bone marrow cells were plated in the presence of 200 ⁇ l of conditioned medium in a soft agar culture in 1 ml final volume. Colonies were scored on day 7 after plating, and the results are shown in Figure 7. The colonies were stained and further typed depending on cell morphology. The results are summarised in Table 3.
- CSA colony stimulating activity
- Conditioned medium was harvested from irradiated stromal cell cultures derived from bone marrow of G-CSF/GM-CSF double deficient mice and stimulated with heat killed Candida albi cans .
- 50 ⁇ l of conditioned medium was added to 5,000 metrizamide-fractionated bone marrow cells in a total volume of 200 ⁇ l in 96 well round-bottomed plates and incubated for 96 h at 37°C in a humidified atmosphere containing 5% C0 2 .
- Cells were also incubated with M-CSF, SCF, IL-3, or medium alone under identical conditions. Parallel cultures were also established in 16 well chamber slides.
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Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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AU60831/98A AU728977B2 (en) | 1997-03-04 | 1998-03-04 | Models for haematopoiesis |
EP98905158A EP0981603A4 (en) | 1997-03-04 | 1998-03-04 | Models for haematopoiesis |
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AUPO5409A AUPO540997A0 (en) | 1997-03-04 | 1997-03-04 | Models for haematopoiesis |
AUPO5409 | 1997-03-04 |
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WO1998039413A1 true WO1998039413A1 (en) | 1998-09-11 |
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PCT/AU1998/000139 WO1998039413A1 (en) | 1997-03-04 | 1998-03-04 | Models for haematopoiesis |
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EP (1) | EP0981603A4 (en) |
AU (1) | AUPO540997A0 (en) |
WO (1) | WO1998039413A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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EP2044197A1 (en) * | 2006-07-24 | 2009-04-08 | The University of Queensland | Method of producing a population of cells |
Citations (1)
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AU6255894A (en) * | 1994-03-04 | 1995-09-18 | Ludwig Institute For Cancer Research | Animals with targeted gene disruption |
Family Cites Families (1)
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US5677139A (en) * | 1995-04-21 | 1997-10-14 | President And Fellows Of Harvard College | In vitro differentiation of CD34+ progenitor cells into T lymphocytes |
-
1997
- 1997-03-04 AU AUPO5409A patent/AUPO540997A0/en not_active Abandoned
-
1998
- 1998-03-04 WO PCT/AU1998/000139 patent/WO1998039413A1/en not_active Application Discontinuation
- 1998-03-04 EP EP98905158A patent/EP0981603A4/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU6255894A (en) * | 1994-03-04 | 1995-09-18 | Ludwig Institute For Cancer Research | Animals with targeted gene disruption |
Non-Patent Citations (9)
Title |
---|
LIESCHKE G., "CSF Deficient Mice - What Have They Taught Us?", CIBA FOUNDATION SYMPOSIUM, 1997, 204, pages 60-77. * |
LIESCHKE G., DUNN A., "Granulocyte Colony-Stimulating Factor (G-CSF) - Deficient Mice", 1998, Ch. 23, CONTEMPORARY IMMUNOLOGY. 1. CYTOKINE KNOCKOUTS, Ed. DURUM SK., MUEGGE K., HUMANA PRESS, TOTOWA, NJ, USA, pp. 440-441. * |
LIESCHKE G., GRAIL D., HODGSON G., METCALF D., STANLEY E. et al., "Mice Lacking Role of Granulocyte Colony Stimulating Factor....", BLOOD, 1994, 84(8), pages 1737-1746. * |
METCALF D., ROBB L., DUNN A.R., MIFSUD S., DI RAGO L., "Role of Granulocyte-Macrophage Colony Stimulating Factor....", BLOOD, 1996, 88(19), pages 3755-3764. * |
NAKANO T., "In Vitro Development of Haematopoietic System....", INT. J. HAEMATOL., 1996, 65, pages 1-8. * |
NAKANO T., "Lymphohaematopoietic Development from Embryonic Stem Cells", SEMINARS IN IMMUNOLOGY, 1995, 7(3), pages 197-203. * |
NISHINAKAMURA R., MYAJIMA P., MEE J., TYBULEWICZ V., MURRAY R., "Haematopoiesis in Mice Lacking the Entire....", BLOOD, 1996, 88(7), pages 2458-2484. * |
See also references of EP0981603A4 * |
ZHAN Y., LIESCHKE G., GRAIL D., DUNN A., CHEERS C., "Essential Roles for Granulocyte-Macrophage Colony Stimulating Factor....", BLOOD, 1998, 91(3), pages 863-9. * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2044197A1 (en) * | 2006-07-24 | 2009-04-08 | The University of Queensland | Method of producing a population of cells |
EP2044197A4 (en) * | 2006-07-24 | 2011-01-05 | Univ Queensland | Method of producing a population of cells |
Also Published As
Publication number | Publication date |
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AUPO540997A0 (en) | 1997-03-27 |
EP0981603A4 (en) | 2003-01-02 |
EP0981603A1 (en) | 2000-03-01 |
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