CA2358146A1 - A method of inhibiting immunologically mediated abortions - Google Patents
A method of inhibiting immunologically mediated abortions Download PDFInfo
- Publication number
- CA2358146A1 CA2358146A1 CA 2358146 CA2358146A CA2358146A1 CA 2358146 A1 CA2358146 A1 CA 2358146A1 CA 2358146 CA2358146 CA 2358146 CA 2358146 A CA2358146 A CA 2358146A CA 2358146 A1 CA2358146 A1 CA 2358146A1
- Authority
- CA
- Canada
- Prior art keywords
- cells
- apoptotic
- leukocytes
- paternal
- induced
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000002401 inhibitory effect Effects 0.000 title abstract description 5
- 206010000210 abortion Diseases 0.000 title description 18
- 231100000176 abortion Toxicity 0.000 title description 17
- 230000001404 mediated effect Effects 0.000 title description 5
- 230000001640 apoptogenic effect Effects 0.000 claims abstract description 37
- 210000000265 leukocyte Anatomy 0.000 claims abstract description 34
- 230000008775 paternal effect Effects 0.000 claims abstract description 21
- 208000000995 spontaneous abortion Diseases 0.000 claims abstract description 16
- 206010000234 Abortion spontaneous Diseases 0.000 claims abstract description 15
- 230000008774 maternal effect Effects 0.000 claims abstract description 12
- 241000124008 Mammalia Species 0.000 claims abstract description 6
- 230000000306 recurrent effect Effects 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 27
- 230000035935 pregnancy Effects 0.000 claims description 19
- 230000006907 apoptotic process Effects 0.000 claims description 16
- 235000003642 hunger Nutrition 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 9
- 230000037351 starvation Effects 0.000 claims description 9
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 4
- 210000004443 dendritic cell Anatomy 0.000 claims description 3
- 210000003719 b-lymphocyte Anatomy 0.000 claims 2
- 210000001616 monocyte Anatomy 0.000 claims 2
- 210000000440 neutrophil Anatomy 0.000 claims 2
- 101100255205 Caenorhabditis elegans rsa-2 gene Proteins 0.000 claims 1
- 238000004132 cross linking Methods 0.000 claims 1
- 230000001939 inductive effect Effects 0.000 claims 1
- 230000006698 induction Effects 0.000 abstract description 2
- 238000002347 injection Methods 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- 239000002158 endotoxin Substances 0.000 description 13
- 230000028993 immune response Effects 0.000 description 13
- 229920006008 lipopolysaccharide Polymers 0.000 description 13
- 210000004698 lymphocyte Anatomy 0.000 description 13
- 231100000673 dose–response relationship Toxicity 0.000 description 12
- 230000005764 inhibitory process Effects 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 238000001802 infusion Methods 0.000 description 8
- 102000004127 Cytokines Human genes 0.000 description 7
- 108090000695 Cytokines Proteins 0.000 description 7
- 238000011752 CBA/J (JAX™ mouse strain) Methods 0.000 description 5
- 230000028709 inflammatory response Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 210000004988 splenocyte Anatomy 0.000 description 5
- 238000011765 DBA/2 mouse Methods 0.000 description 4
- 230000000056 copulatory effect Effects 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 230000017074 necrotic cell death Effects 0.000 description 4
- 210000003462 vein Anatomy 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 102000003814 Interleukin-10 Human genes 0.000 description 3
- 108090000174 Interleukin-10 Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000222722 Leishmania <genus> Species 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 229940123573 Protein synthesis inhibitor Drugs 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000001338 necrotic effect Effects 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000007 protein synthesis inhibitor Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000036962 time dependent Effects 0.000 description 2
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 206010003497 Asphyxia Diseases 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 206010020843 Hyperthermia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000013564 activation of immune response Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000005975 antitumor immune response Effects 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000036031 hyperthermia Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006882 induction of apoptosis Effects 0.000 description 1
- 230000006749 inflammatory damage Effects 0.000 description 1
- 210000005061 intracellular organelle Anatomy 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000029069 type 2 immune response Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/06—Antiabortive agents; Labour repressants
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Zoology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Endocrinology (AREA)
- Gynecology & Obstetrics (AREA)
Abstract
The invention disclosed teaches methods of inhibiting recurrent spontaneous abortion in a mammal through administration of apoptotic leukocytes. Said leukocytes may be derived from maternal or paternal sources. Several methods of apoptotic induction may be used to attain anti-abortogenic erects.
Description
DISCLOSURE
Field of the Invention The invention teaches methods of modulating the immune system of a mammal in order to prevent spontaneous abortion.
Background Recurrent Spontaneous Abortion Recurrent Spontaneous Abortion (RSA) affects 1-2% of women in North America (1). RSA is believed to be caused by abnormal activation of Thl cells, these cells are generally suppressed in pregnancy. Lin et al demonstrated successful murine pregnancy is associated with upregulated production of the Th2 cytokines IL-4, IL-5 and II,-10 in the fetoplacental unit (2). This was postulated to inhibit development of Thl cells, cells associated with induction of inflammatory responses. The Th2 biasing of immune response by pregnancy can be seen in C57BL/6 mice infected with Leishmania. Normally C57BL/6 mice are resistant to such infection because of a Thl bias. When these mice are pregnant, due to the Th2 biasing of pregnancy, infection with Leishmania is possible (3).
Administration of Thl cytokines to pregnant mice can increase the rate of abortion (4), whereas administration of Th2 cytokine suppresses it (5).
Therapies which prevent RSA, such as paternal lymphocyte infusion are associated with augmentation of Th2 responses (6,7). Therefore it appears that successful pregnancy requires suppression of Thl and promotion of Th2 immune responses.
Paternal lymphocyte infusion therapy was originally devised by Govallo based on the idea that RSA occurs due to improper recognition of paternal antigens by maternal immune system cells (8). Through immunization with paternal lymphocytes, the mother's immune system will be primed to recognize the paternal antigens on the placenta, thus immune response will occur. However, Govallo believed that stimulation of antiplacental immune response by injection of paternal lymphocytes would not cause an inflammatory response, but an immune response which would actually help the placenta to grow. A parallel concept was introduced by Prehn in cancer immunology who should that host antitumor immune response in some circumstance can promote tumor growth (9).
Although some clinical trials using paternal lymphocyte infusion have reported a decreased number of RSA (10,11), a recent double blind study reported no difference between treated and control groups (12). Nevertheless this practice is still in use today.
Rational exists for the systemic administration of Th2 cytokines in women prone to RSA. Since cytokine like IL-10 can be safely administered, and prevent activation of Thl immune responses (13). The problem with systemic cytokine therapy is cost associated, as well as the possibility of global immune suppression.
Thus there is a need for treatment of RSA in an economical, safe and effective manner.
Apoptosis Apoptosis, in contrast to necrosis, is a type of cell death in which membrane integrity of the cell is kept, thus preventing leakage of intracellular organelles to the extracellular mileau (14). The process of apoptosis exists in order to rid the organism of unwanted cells without causing an inflammatory response. In contrast, cells dying by necrosis release various intracellular components causing inflammation and tissue damage. The concept of "danger signals" was originally proposed by Matzinger (15) with the idea that innate signals of danger exist, which instruct the body to launch immune response or to ignore the antigen.
Under Matzinger's paradigm, danger is associated with tissue necrosis, whereas lack of danger occurs when cells die by apoptosis. This is exemplified by studies showing that intracellular danger signals are released by cells undergoing necrotic but not apoptotic cell death. Such danger signals include free-floating heat shock proteins which can activate dendritic cells and macrophages ( 16, 17).
Following with the idea that cells undergoing necrosis cause immune activation through the danger pathway, apoptotic death should inhibit activation of immune response.
One method by which apoptotic cells inhibit immune response is through the production of immunosuppressive cytokines such as II,-10. In a study by Gao et al, T cells induced to undergo apoptosis secreted IL-10 protein, as well as increased production of IL-10 transcripts. In the same study it was demonstrated that apoptotic T cells can program macrophages to inhibit activation of pro-inflammatory Thl immune responses (18). In another study, Voll et al demonstrated that apoptotic lymphocytes can suppress production of 1'NF-oc and IL-12 from peripheral blood mononuclear cells activated with lipopolysaccharide (LPS) (19).
Conclusion In light of observations that paternal lymphocytes infusions seem to diminish spontaneous abortion, and that lymphocytes undergoing apoptosis inhibit inflammatory responses, we investigated whether transfer of paternal and maternal origin apoptotic lymphocytes into abortion-prone mice would protect the fetus from immunologically mediated destruction.
Summary of the Invention The disclosed invention teaches a method of inhibiting abortogenic immune responses through administration of treated blood cells into a pregnant mammal.
More specifically, paternal or maternal leukocytes are treated with apoptotic stimuli after which they are introduced into a pregnant female. This process, we postulate, results in the inhibition of inflammatory immune responses, allowing for completion of pregnancy in situations under which the immune response would normally result in fetal destruction.
The invention discloses methods of preparing paternal or maternal leukocytes, in a manner which endows the cells with anti-inflammatory properties. Disclosed is the utility of these cells in inhibiting anti-fetal immune responses. The direct effect of administration of the treated leukocytes on the pregnant female, that is the anti-abortogenic effect, is also disclosed.
Brief Description of the Drawings Figure I illustrates time-dependent apoptotic effects of serum starvation on murine leukocytes.
Figure II illustrates the dose-dependent antiabortogenic effects of infusing apoptotic paternal cells to a pregnant mother induced to undergo abortion by LPS
administration.
Figure III illustrates the dose-dependent antiabortogenic effects of infusing apoptotic maternal cells to a pregnant mother induced to undergo abortion by LPS
administration.
Figure IV illustrates the dose-dependent antiabortogenic effects of infusing paternal apoptotic cells into the CBA/J X DBAJ2 model of spontaneous abortion.
Figure V illustrates the dose-dependent antiabortogenic effects of infusing maternal apoptotic cells into the CBA/J X DBA/2 model of spontaneous abortion.
Detailed Description of Preferred Embodiments This invention teaches methods of preventing immunologically-mediated spontaneous abortions through administration of apoptotic lymphocytes. This effect, we believe is mediated through production of soluble and membrane bound components on apoptotic cells which suppress inflammatory immune responses.
It is these inflammatory responses which bring about the demise of the conceptus.
In the data displayed apoptosis was achieved through serum starvation of the leukocytes. In other studies we performed similar antiabortigenic effects can be seen by injection of leukocytes in which apoptosis was induced through other methods such as irradiation or treatment with protein synthesis inhibitors. In agreement with previously published studies, the immunomodulatory effects of apoptotic leukocytes are independent of the method by which apoptosis was induced (18, 19).
One embodiment of the invention mends itself applicable to the current practice of immunizing pregnant women at risk of spontaneous abortion with paternal lymphocytes. Paternal leukocytes can be purified from whole blood of the father through the FicollTM technique or other methods known to one skilled in the art.
Apoptosis can be induced in these cells through serum starvation or by other methods known such as irradiation, treatment with protein synthesis inhibitors, treatment with ozone gas, or treatment with hyperthermia. Quality of the apoptotic cells can be assessed by Annexin-V staining as well as propidium iodide in order to ensure a high percentage of apoptotic but not necrotic cells are administered. Injection of apoptotic cells into the mother can occur via direct intravenous administration using syringe and needle, or continuous drip administration. The same procedure can be carried out using leukocytes derived from the mother.
Detection of cytokines associated with spontaneous abortion such as interferon-y can serve as an immune marker to guide the number of apoptotic cells injected, as well as the frequency of injection.
Examples Example I
Induction of Apoptosis by Serum Starvation BALB/c mice were euthanized by carbon dioxide asphyxiation and spleens were collected under asceptic conditions. Spleens were disassociated with a wire mesh and erythrocytes were lysed with hypotonic buffer. Lysed erythrocytes were removed by centrifizgation. Splenocytes were cultured at a concentration of cells/well in serum-free RPMI media in 96 well plates in a total volume of 200 p,1.
At day 0. 1, 2, 3, and 4 apoptosis was assessed using ApoAlertT"' Annexin V-FITC apoptosis kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. As illustrated in Figure I a time dependent increase in apoptotic cells was observed.
Example II
Dose Dependent Inhibition of LPS-Induced Spontaneous Abortion by Infusion of Apoptotic Paternal Leukocytes Pregnancy was induced in 8 week old BALB/c females by crossing with C57B6 males. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 of pregnancy a single injection of 4 p,g lipopolysaccharide from Escherichia coli (LPS: Sigma Chemicals, St. Louis, MO), was administered in 100 p1 of saline through the intraperitoneal route. On day 5 an injection of apoptotic paternal (C57B6) leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure II administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
Example III
Dose Dependent Inhibition of LPS Induced Spontaneous Abortion by Infusion of Apoptotic Maternal Leukocytes Pregnancy was induced in 8 week old BALB/c females by crossing with C57/B6 males. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 of pregnancy a single injection of 4 p,g lipopolysaccharide from Escherichia coli (LPS: Sigma Chemicals, St. Louis, MO), was administered in 100 p,1 of saline through the intraperitoneal route. On day 5 an injection of apoptotic maternal (BALB/c) leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure III administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
Example IV
Dose-Dependent Inhibition Spontaneous Abortion in The CBAl,I X DBAl2 Model by Infusion of Apoptotic Paternal Lymphocytes.
CBA/J female mice were bred with DBA/2. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 an injection of apoptotic paternal (DBA/2) leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure IV administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
Example V
Dose Dependent Inhibition Spontaneous Abortion in The CBAlJX DBAl2 Model by Infusion of Apoptotic Maternal Lymphocytes.
CBA/J female mice were bred with DBA/2. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 an injection of apoptotic maternal (CBA/J~ leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure V administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
The invention discloses a method of inhibiting immunologically mediated abortions in a mammal. It is to be understood that the above examples are presented only for clarity and that the invention may take other embodiments as practiced by one skilled in the art.
References:
1. Coulman CB, Page WP, McIntyre JA. Overview of recurrent pregnancy loss.
Immunological Obstetrics. Norton & Co, New York London, pp 333-356, 1992.
Field of the Invention The invention teaches methods of modulating the immune system of a mammal in order to prevent spontaneous abortion.
Background Recurrent Spontaneous Abortion Recurrent Spontaneous Abortion (RSA) affects 1-2% of women in North America (1). RSA is believed to be caused by abnormal activation of Thl cells, these cells are generally suppressed in pregnancy. Lin et al demonstrated successful murine pregnancy is associated with upregulated production of the Th2 cytokines IL-4, IL-5 and II,-10 in the fetoplacental unit (2). This was postulated to inhibit development of Thl cells, cells associated with induction of inflammatory responses. The Th2 biasing of immune response by pregnancy can be seen in C57BL/6 mice infected with Leishmania. Normally C57BL/6 mice are resistant to such infection because of a Thl bias. When these mice are pregnant, due to the Th2 biasing of pregnancy, infection with Leishmania is possible (3).
Administration of Thl cytokines to pregnant mice can increase the rate of abortion (4), whereas administration of Th2 cytokine suppresses it (5).
Therapies which prevent RSA, such as paternal lymphocyte infusion are associated with augmentation of Th2 responses (6,7). Therefore it appears that successful pregnancy requires suppression of Thl and promotion of Th2 immune responses.
Paternal lymphocyte infusion therapy was originally devised by Govallo based on the idea that RSA occurs due to improper recognition of paternal antigens by maternal immune system cells (8). Through immunization with paternal lymphocytes, the mother's immune system will be primed to recognize the paternal antigens on the placenta, thus immune response will occur. However, Govallo believed that stimulation of antiplacental immune response by injection of paternal lymphocytes would not cause an inflammatory response, but an immune response which would actually help the placenta to grow. A parallel concept was introduced by Prehn in cancer immunology who should that host antitumor immune response in some circumstance can promote tumor growth (9).
Although some clinical trials using paternal lymphocyte infusion have reported a decreased number of RSA (10,11), a recent double blind study reported no difference between treated and control groups (12). Nevertheless this practice is still in use today.
Rational exists for the systemic administration of Th2 cytokines in women prone to RSA. Since cytokine like IL-10 can be safely administered, and prevent activation of Thl immune responses (13). The problem with systemic cytokine therapy is cost associated, as well as the possibility of global immune suppression.
Thus there is a need for treatment of RSA in an economical, safe and effective manner.
Apoptosis Apoptosis, in contrast to necrosis, is a type of cell death in which membrane integrity of the cell is kept, thus preventing leakage of intracellular organelles to the extracellular mileau (14). The process of apoptosis exists in order to rid the organism of unwanted cells without causing an inflammatory response. In contrast, cells dying by necrosis release various intracellular components causing inflammation and tissue damage. The concept of "danger signals" was originally proposed by Matzinger (15) with the idea that innate signals of danger exist, which instruct the body to launch immune response or to ignore the antigen.
Under Matzinger's paradigm, danger is associated with tissue necrosis, whereas lack of danger occurs when cells die by apoptosis. This is exemplified by studies showing that intracellular danger signals are released by cells undergoing necrotic but not apoptotic cell death. Such danger signals include free-floating heat shock proteins which can activate dendritic cells and macrophages ( 16, 17).
Following with the idea that cells undergoing necrosis cause immune activation through the danger pathway, apoptotic death should inhibit activation of immune response.
One method by which apoptotic cells inhibit immune response is through the production of immunosuppressive cytokines such as II,-10. In a study by Gao et al, T cells induced to undergo apoptosis secreted IL-10 protein, as well as increased production of IL-10 transcripts. In the same study it was demonstrated that apoptotic T cells can program macrophages to inhibit activation of pro-inflammatory Thl immune responses (18). In another study, Voll et al demonstrated that apoptotic lymphocytes can suppress production of 1'NF-oc and IL-12 from peripheral blood mononuclear cells activated with lipopolysaccharide (LPS) (19).
Conclusion In light of observations that paternal lymphocytes infusions seem to diminish spontaneous abortion, and that lymphocytes undergoing apoptosis inhibit inflammatory responses, we investigated whether transfer of paternal and maternal origin apoptotic lymphocytes into abortion-prone mice would protect the fetus from immunologically mediated destruction.
Summary of the Invention The disclosed invention teaches a method of inhibiting abortogenic immune responses through administration of treated blood cells into a pregnant mammal.
More specifically, paternal or maternal leukocytes are treated with apoptotic stimuli after which they are introduced into a pregnant female. This process, we postulate, results in the inhibition of inflammatory immune responses, allowing for completion of pregnancy in situations under which the immune response would normally result in fetal destruction.
The invention discloses methods of preparing paternal or maternal leukocytes, in a manner which endows the cells with anti-inflammatory properties. Disclosed is the utility of these cells in inhibiting anti-fetal immune responses. The direct effect of administration of the treated leukocytes on the pregnant female, that is the anti-abortogenic effect, is also disclosed.
Brief Description of the Drawings Figure I illustrates time-dependent apoptotic effects of serum starvation on murine leukocytes.
Figure II illustrates the dose-dependent antiabortogenic effects of infusing apoptotic paternal cells to a pregnant mother induced to undergo abortion by LPS
administration.
Figure III illustrates the dose-dependent antiabortogenic effects of infusing apoptotic maternal cells to a pregnant mother induced to undergo abortion by LPS
administration.
Figure IV illustrates the dose-dependent antiabortogenic effects of infusing paternal apoptotic cells into the CBA/J X DBAJ2 model of spontaneous abortion.
Figure V illustrates the dose-dependent antiabortogenic effects of infusing maternal apoptotic cells into the CBA/J X DBA/2 model of spontaneous abortion.
Detailed Description of Preferred Embodiments This invention teaches methods of preventing immunologically-mediated spontaneous abortions through administration of apoptotic lymphocytes. This effect, we believe is mediated through production of soluble and membrane bound components on apoptotic cells which suppress inflammatory immune responses.
It is these inflammatory responses which bring about the demise of the conceptus.
In the data displayed apoptosis was achieved through serum starvation of the leukocytes. In other studies we performed similar antiabortigenic effects can be seen by injection of leukocytes in which apoptosis was induced through other methods such as irradiation or treatment with protein synthesis inhibitors. In agreement with previously published studies, the immunomodulatory effects of apoptotic leukocytes are independent of the method by which apoptosis was induced (18, 19).
One embodiment of the invention mends itself applicable to the current practice of immunizing pregnant women at risk of spontaneous abortion with paternal lymphocytes. Paternal leukocytes can be purified from whole blood of the father through the FicollTM technique or other methods known to one skilled in the art.
Apoptosis can be induced in these cells through serum starvation or by other methods known such as irradiation, treatment with protein synthesis inhibitors, treatment with ozone gas, or treatment with hyperthermia. Quality of the apoptotic cells can be assessed by Annexin-V staining as well as propidium iodide in order to ensure a high percentage of apoptotic but not necrotic cells are administered. Injection of apoptotic cells into the mother can occur via direct intravenous administration using syringe and needle, or continuous drip administration. The same procedure can be carried out using leukocytes derived from the mother.
Detection of cytokines associated with spontaneous abortion such as interferon-y can serve as an immune marker to guide the number of apoptotic cells injected, as well as the frequency of injection.
Examples Example I
Induction of Apoptosis by Serum Starvation BALB/c mice were euthanized by carbon dioxide asphyxiation and spleens were collected under asceptic conditions. Spleens were disassociated with a wire mesh and erythrocytes were lysed with hypotonic buffer. Lysed erythrocytes were removed by centrifizgation. Splenocytes were cultured at a concentration of cells/well in serum-free RPMI media in 96 well plates in a total volume of 200 p,1.
At day 0. 1, 2, 3, and 4 apoptosis was assessed using ApoAlertT"' Annexin V-FITC apoptosis kit (Clontech, Palo Alto, CA) according to the manufacturer's instructions. As illustrated in Figure I a time dependent increase in apoptotic cells was observed.
Example II
Dose Dependent Inhibition of LPS-Induced Spontaneous Abortion by Infusion of Apoptotic Paternal Leukocytes Pregnancy was induced in 8 week old BALB/c females by crossing with C57B6 males. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 of pregnancy a single injection of 4 p,g lipopolysaccharide from Escherichia coli (LPS: Sigma Chemicals, St. Louis, MO), was administered in 100 p1 of saline through the intraperitoneal route. On day 5 an injection of apoptotic paternal (C57B6) leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure II administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
Example III
Dose Dependent Inhibition of LPS Induced Spontaneous Abortion by Infusion of Apoptotic Maternal Leukocytes Pregnancy was induced in 8 week old BALB/c females by crossing with C57/B6 males. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 of pregnancy a single injection of 4 p,g lipopolysaccharide from Escherichia coli (LPS: Sigma Chemicals, St. Louis, MO), was administered in 100 p,1 of saline through the intraperitoneal route. On day 5 an injection of apoptotic maternal (BALB/c) leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure III administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
Example IV
Dose-Dependent Inhibition Spontaneous Abortion in The CBAl,I X DBAl2 Model by Infusion of Apoptotic Paternal Lymphocytes.
CBA/J female mice were bred with DBA/2. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 an injection of apoptotic paternal (DBA/2) leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure IV administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
Example V
Dose Dependent Inhibition Spontaneous Abortion in The CBAlJX DBAl2 Model by Infusion of Apoptotic Maternal Lymphocytes.
CBA/J female mice were bred with DBA/2. Day zero of pregnancy was established as the day copulatory plug was observed. On day 5 an injection of apoptotic maternal (CBA/J~ leukocytes was administered intravenously through the tail vein. Apoptotic leukocytes were induced by serum starvation for 4 days as described in Example I. Mice were sacrificed on day 12 of pregnancy. As shown in Figure V administration of fresh splenocytes (Untreated leukocytes) did not inhibit LPS-induced abortions whereas injection of apoptotic leukocytes induced a dose-dependent inhibition of abortions. Abortions were quantified as percent resorption identified by morphological appearance. Each bar represents the average (~Standard Deviation) of 7 mice.
The invention discloses a method of inhibiting immunologically mediated abortions in a mammal. It is to be understood that the above examples are presented only for clarity and that the invention may take other embodiments as practiced by one skilled in the art.
References:
1. Coulman CB, Page WP, McIntyre JA. Overview of recurrent pregnancy loss.
Immunological Obstetrics. Norton & Co, New York London, pp 333-356, 1992.
2. Lin H, Mosmann TR, Guilbert L, Tuntipopipat S, Wegmann TG. Synthesis of T helper 2-type cytokines at the maternal-fetal interface. J Immunol. 1993 Nov 1;151 (9):4562-73.
3. Krishnan L, Guilbert LJ, Russell AS, Wegmann TG, Mosmann TR, Belosevic M. Pregnancy impairs resistance of C57BL/6 mice to Leishmania major infection and causes decreased antigen-specific lFN-gamma response and increased production of T helper 2 cytokines. J Immunol. 1996 Jan 15;156(2):644-52.
4. Chaouat G, Menu E, Clark DA, Dy M, Minkowski M, Wegmann TG. Control of fetal survival in CBA x DBA/2 mice by lymphokine therapy. J Reprod Fertil.
1990 Ju1;89(2):447-58.
1990 Ju1;89(2):447-58.
5. Chaouat G, Assal Meliani A, Martal J, Raghupathy R, Elliot J, Mosmann T, Wegmann TG. IL-10 prevents naturally occurring fetal loss in the CBA x DBA/2 mating combination, and local defect in II,-10 production in this abortion-prone combination is corrected by in vivo injection of IFN-tau. J Immunol. 1995 May 1;154(9):4261-8.
6. Szpakowski A, Malinowski A, Glowacka E, Wilczynski JR, Kolasa D, Dynski M, Tchorzewski H, Zeman K, Szpakowski M. The influence of paternal lymphocyte immunization on the balance of Thl/Th2 type reactivity in women with unexplained recurrent spontaneous abortion. Ginekol Pol. 2000 Jun;71(6):586-92.
7. Hayakawa S, Karasaki-Suzuki M, Itoh T, Ishii M, Kanaeda T, Nagai N, Takahashi-Yamamoto N, Tochigi M, Chishima F, Fujii TK, Oyama J, Kitanaka S, Satoh K. Effects of paternal lymphocyte immunization on peripheral Thl/Th2 balance and TCR V beta and V gamma repertoire usage of patients with recurrent spontaneous abortions. Am J Reprod Immunol. 2000 Feb;43(2):107-1S.
8. Govallo VI, Sidel'nikov VM. Immunization of pregnant women with allogenic lymphocytes from the husband as a method of preventing spontaneous abortion.
Akush Ginekol (Mosk). 1983 Dec;(12):2S-8.
Akush Ginekol (Mosk). 1983 Dec;(12):2S-8.
9. Prehn RT. Immunostimulation of the lymphodependent phase of neoplastic growth.
J Natl Cancer Inst. 1977 Oct;S9(4):1043-9.
J Natl Cancer Inst. 1977 Oct;S9(4):1043-9.
10. Gatenby PA, Cameron K, Simes RJ, Adelstein S, Bennett MJ, Jansen RP, Shearman RP, Stewart GJ, Whittle M, Doran TJ. Treatment of recurrent spontaneous abortion by immunization with paternal lymphocytes: results of a controlled trial. Am J Reprod Immunol. 1993 Mar;29(2):88-94.
11. Mowbray JF, Gibbings C, Liddell H, Reginald PW, Underwood JL, Beard RW.
Controlled trial of treatment of recurrent spontaneous abortion by immunisation with paternal cells. Lancet. 1985 Apr 27;1(8435):941-3.
Controlled trial of treatment of recurrent spontaneous abortion by immunisation with paternal cells. Lancet. 1985 Apr 27;1(8435):941-3.
12. Ober C, Karnson T, Odem RR, Barnes RB, Branch DW, Stephenson MD, Baron B, Walker MA, Scott JR, Schreiber JR. Mononuclear-cell immunisation in prevention of recurrent miscarriages: a randomised trial. Lancet. 1999 Jul 31;354(9176):365-9.
13. Reich K, Garbe C, Blaschke V, Maurer C, Middel P, Westphal G, Lippert U, Neumann C. Response of psoriasis to interleukin-10 is associated with suppression of cutaneous type 1 inflammation, downregulation of the epidermal interleukin-8/CXCR2 pathway and normalization of keratinocyte maturation. J
Invest Dermatol. 2001 Feb;116(2):319-29.
Invest Dermatol. 2001 Feb;116(2):319-29.
14. Kerr, J. F. R., Wyllie, A. H. & Curne, A. R. Apoptosis: a basic biological phenomenon with widespread implications in tissue kinetics. Br. J. Cancer 1972, 26, 239-257.
15. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol. 1994;12:991-1045.
16. Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK. Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol. 2000 Nov;l2(l 1):1539-46.
17. Basu S, Binder RJ, Ramalingam T, Srivastava PK. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin.
Immunity.
2001 Mar;l4(3):303-13.
Immunity.
2001 Mar;l4(3):303-13.
18. Gao Y, Herndon JM, Zhang H, Gri~th TS, Ferguson TA. Antiinflammatory effects of CD95 ligand (FasL)-induced apoptosis. J Exp Med. 1998 Sep 7;188(5):887-96.
19. Voll G, Zhong J, Block M Immunosuppressive aspects of apoptotic peripheral blood mononuclear cells. Nature 390:350, 1997
Claims (6)
1. A method of preventing recurrent spontaneous abortion in a mammal through (a) Withdrawing leukocytes from a donor (b) Inducing apoptosis of the donor leukocytes (c) Administration of the donor leukocytes to a female susceptible to RSA
2. The method of claim I in which leukocyte defines a purified population of neutrophils, monocytes, dendritic cells, T cells or B cells.
3. The method of claim I in which leukocyte defines a combination of neutrophils, monocytes, dendritic cells, T cells and B cells.
4. The method of claim I where apoptosis is induced through ultraviolet irradiation, X-irradiation, .gamma.-irradiation, serum starvation or crosslinking of Fas.
5. The method of claim I where the apoptotic cells are used to prevent recurrent spontaneous abortion in a mammal.
6. The method of claim I where the donor can be maternal or paternal in reference to the pregnancy.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2358146 CA2358146A1 (en) | 2001-09-19 | 2001-09-19 | A method of inhibiting immunologically mediated abortions |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2358146 CA2358146A1 (en) | 2001-09-19 | 2001-09-19 | A method of inhibiting immunologically mediated abortions |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2358146A1 true CA2358146A1 (en) | 2003-03-19 |
Family
ID=4170155
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2358146 Abandoned CA2358146A1 (en) | 2001-09-19 | 2001-09-19 | A method of inhibiting immunologically mediated abortions |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2358146A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1719516A1 (en) * | 2004-02-20 | 2006-11-08 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A pharmaceutical composition used for treating recurrent spontaneous abortion and method thereof |
-
2001
- 2001-09-19 CA CA 2358146 patent/CA2358146A1/en not_active Abandoned
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1719516A1 (en) * | 2004-02-20 | 2006-11-08 | Beijing Xinjing Antai Medical And Technology Service Limited Corp. | A pharmaceutical composition used for treating recurrent spontaneous abortion and method thereof |
| EP1719516A4 (en) * | 2004-02-20 | 2007-03-28 | Beijing Xinjing Antai Medical | A pharmaceutical composition used for treating recurrent spontaneous abortion and method thereof |
| US7902162B2 (en) | 2004-02-20 | 2011-03-08 | Beijin Xinjing Antai Medical and Technology Service Limited Corp | Medicament and method for treating recurrent spontaneous abortion |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6553219B2 (en) | Methods and compositions for tumor clearing | |
| Sabel | Cryo-immunology: a review of the literature and proposed mechanisms for stimulatory versus suppressive immune responses | |
| Dunn‐Pirio et al. | Immunotherapy approaches in the treatment of malignant brain tumors | |
| Bronte et al. | Apoptotic death of CD8+ T lymphocytes after immunization: induction of a suppressive population of Mac-1+/Gr-1+ cells | |
| Baskar et al. | Major histocompatibility complex class II+ B7-1+ tumor cells are potent vaccines for stimulating tumor rejection in tumor-bearing mice. | |
| Fuchs et al. | Is cancer dangerous to the immune system? | |
| Grabbe et al. | Tumor antigen presentation by epidermal antigen‐presenting cells in the mouse: modulation by granulocyte‐macrophage colony‐stimulating factor, tumor necrosis factor α, and ultraviolet radiation | |
| Nikitina et al. | Combination of γ‐irradiation and dendritic cell administration induces a potent antitumor response in tumor‐bearing mice: approach to treatment of advanced stage cancer | |
| Peng et al. | T-cell adoptive therapy of tumors: mechanisms of improved therapeutic performance | |
| EP1912672B1 (en) | Defective ribosomal products in blebs (dribbles) and methods of use to stimulate an immune response | |
| JP2000515127A (en) | GM-CSF administration for treatment and prevention of brain tumor recurrence | |
| CN103648524A (en) | Means and methods for active cellular immunotherapy of cancer by using tumor cells killed by high hydrostatic pressure and dendritic cells | |
| Hongo et al. | Tolerogenic interactions between CD8+ dendritic cells and NKT cells prevent rejection of bone marrow and organ grafts | |
| JP2017141287A (en) | Induction of IL-12 using immunotherapy | |
| JP2008523067A (en) | Alpha thymosin peptides as cancer vaccine adjuvants | |
| Azuma et al. | Use of lymphokine-activated killer cells to prevent bone marrow graft rejection and lethal graft-vs-host disease. | |
| EP1670513A2 (en) | Use of cox-2 inhibitor to prevent t-cell anergy induced by dendritic cell therapy | |
| CA2358146A1 (en) | A method of inhibiting immunologically mediated abortions | |
| Bassiony et al. | Immunotherapy and cancer | |
| Aloysius et al. | Therapy and host defences | |
| US20040228925A1 (en) | Method for inducing an anti-tumor and anti-cachexia immune response in mammals | |
| Heald et al. | T‐cell responses in photoimmune therapy | |
| KR100522526B1 (en) | Method of Preparing Dendritic cell for Immunotherapy | |
| WO2024085085A1 (en) | Method for producing dendritic cell formulation including dendritic cells that stimulate nkt cells | |
| Jayalie et al. | Combination of Adoptive Cell Therapy and Radiotherapy in Cancer Management |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20040920 |