Methods for treating and preventing infectious disease

a technology of infectious diseases and methods, applied in the field of oligonucleotides, can solve problems such as poly(i,c), and achieve the effect of increasing the sensitivity of chronic leukemia cells

Inactive Publication Date: 2005-05-12
UNIV OF IOWA RES FOUND +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022] Further, the ability of the nucleic acid sequences of the invention described herein to induce leukemic cells to enter the cell cycle supports their use in treating leukemia by increasing the sensitivity of chronic leukemia cells followed by conventional ablative chemotherapy, or by combining the nucleic acid sequences with other immunotherapies.

Problems solved by technology

Unfortunately, toxic side effects have thus far prevented poly (I,C) from becoming a useful therapeutic agent.

Method used

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  • Methods for treating and preventing infectious disease
  • Methods for treating and preventing infectious disease
  • Methods for treating and preventing infectious disease

Examples

Experimental program
Comparison scheme
Effect test

example 1

Effects of ODNs on B Cell Total RNA Synthesis and Cell Cycle

[0152] B cells were purified from spleens obtained from 6-12 week old specific pathogen free DBA / 2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lymphocyte M(Cedarlane Laboratories, Hornby, Ontario, Canada) (“B cells”). B cells contained fewer than 1% CD4+ or CD8+cells. 8×104B cells were dispensed in triplicate into 96 well microtiter plates in 100 μl RPMI containing 10% FBS (heat inactivated to 65° C. for 30 min.), 50 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 ug / ml streptomycin, and 2 mM L-glutamate. 20 μM ODN were added at the start of culture for 20 h at 37° C., cells pulsed with 1 μCi of 3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ALISA spot assay after culture of whole spleen cells with ODN at 20 μM for 48 hr....

example 2

Effects of ODN on Production of IgM from B Cells

[0153] Single cell suspensions form the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting B cells (J. Exp. Med. 155:1523 (1982). These were cultured as described above in 30 μg / ml LPS for 48 hr. The number of B cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D. M. et al. J. Immunol 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labeled anti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St. Louis Mo.) which forms an insoluble blue precipitate in the presence of phosphatase. The dilution of cells producing 20-40 spots / well was used to de...

example 3

B Cell Stimulation by Bacterial DNA

[0154] DBA / 2 B cells were cultured with no DNA or 50 μg / ml of a( Micrococcus lysodeikticus; b) NZB / N mouse spleen; and c) NSF / N mouse spleen genomic DNAs for 48 hours, then pulsed with 3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNASE I for 30 minutes at 37° C. prior to addition to cell cultures. E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISAspot assay.

[0155] DBA / 2 B cells were cultured with either no additive, 50 μg / ml LPS or the ODN 1; la; 4; or 4a at 20 uM. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 μM of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.

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Abstract

Nucleic acid sequences containing unmethylated CpG dinucleotides that modulate an immune response including stimulating a Th1 pattern of immune activation, cytokine production, NK lytic activity, and B cell proliferation are disclosed. The sequences are also useful as a synthetic adjuvant.

Description

RELATED APPLICATION [0001] This application is a continuation of co-pending U.S. Ser. No. 10 / 187,489, filed Jul. 2, 2002, which is a divisional of co-pending U.S. Ser. No. 09 / 630,319, filed Jul. 31, 2000, which is a divisional of U.S. Ser. No. 08 / 960,774, filed Oct. 30, 1997, now issued as U.S. Pat. No. 6,239,116 B1 on May 29, 2001, which is a continuation-in-part of U.S. Ser. No. 08 / 738,652, filed Oct. 30, 1996, now issued as U.S. Pat. No. 6,207,646 B1 on Mar. 27, 2001, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 386,063, filed Feb. 7, 1995, now issued as U.S. Pat. No. 6,194,388 B1 on Feb. 27, 2001, which is a continuation-in-part of U.S. patent application Ser. No. 08 / 276,358, filed Jul. 15, 1994 which is now abandoned, each of which are incorporated herein by reference in their entirety.GOVERNMENT [0002] The work resulting in this invention was supported in part by National Institute of Health Grant No. R29-AR42556-01. The U.S. Government may have right...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09A61K9/127A61K31/00A61K31/175A61K31/335A61K31/47A61K31/4706A61K31/70A61K31/7088A61K39/39A61K39/395A61K45/00A61K47/48A61K48/00A61P1/00A61P1/02A61P1/04A61P11/06A61P17/06A61P19/02A61P31/04A61P31/12A61P33/00A61P35/00A61P37/02A61P37/04A61P37/06A61P37/08A61P43/00C07H21/00C07H21/02C07H21/04C07K14/52C12N15/11C12Q1/68
CPCA61K31/00A61K31/4706A61K31/7048A61K31/711A61K31/7125A61K39/00C12Q1/68A61K2039/55561C07H21/00C12N15/117C12N2310/17C12N2310/315A61K39/39A61P1/00A61P1/02A61P1/04A61P11/06A61P17/06A61P19/02A61P31/04A61P31/12A61P33/00A61P35/00A61P37/02A61P37/04A61P37/06A61P37/08A61P43/00Y02A50/30
Inventor KRIEG, ARTHURKLINMAN, DENNISSTEINBERG, ALFRED
Owner UNIV OF IOWA RES FOUND
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