Exposing lymphocytes to anthrax lethal toxin; contacting the lymphocytes with a test agent; detecting a mitogen-activated protein kinase kinase-dependent cytokine response; increased response or increase in lymphocyte proliferation indicates that the lymphocyte-associated activity is decreased
Inventors
Assignees
US Department of Health and Human Services
Publication Number
US-7803565-B1
Publication Date
2010-09-28
Expiration Date
2026-04-05
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Abstract
It is disclosed herein that isolated lymphocytes, such as human B-cells and CD4+ T-cell can be used to determine an amount of lymphocyte-associated anthrax lethal toxin activity present. Methods of using isolated lymphocytes to identify anthrax therapeutic agents and to determine the efficacy of a potential anthrax therapeutic are disclosed. Methods are also provided for diagnosing and treating anthrax infections.
Core Innovation
The invention discloses methods using isolated lymphocytes, such as human B-cells and CD4+ T cells, to determine the amount of lymphocyte-associated anthrax lethal toxin (LT) activity present. The methods include exposing lymphocytes to anthrax LT, contacting them with a test agent, and detecting a mitogen-activated protein kinase kinase (MAPKK)-dependent cytokine response. An increased cytokine response or increased lymphocyte proliferation indicates decreased LT activity, which suggests the test agent decreases anthrax pathogenicity. These bioassays can also identify therapeutic agents and determine the efficacy of potential anthrax therapeutics.
The disclosed bioassays address limitations in existing anthrax LT activity assays, which rely on species- and strain-specific effects on murine macrophage lines and do not reflect effects in human cells. The invention specifically shows that anthrax LT inhibits MAPKK-dependent cytokine production (such as IL-2, IL-4, and IFN-γ) and lymphocyte proliferation in human lymphocytes. These in vitro assays use human primary or cell line lymphocytes to better correlate with human disease pathology and provide more relevant systems for screening anthrax therapeutics.
The problems solved include the inadequacy of previous assays that depend on murine macrophage cytotoxicity, which is cell-, strain-, and species-specific and not demonstrated in human cells. There is a need for human cell-based bioassays to screen agents that reduce anthrax LT activity and to measure biologically active LT in humans. This invention fills that gap by providing human lymphocyte-based assays that monitor MAPKK-dependent cytokine responses and proliferation as indicators of anthrax LT activity and its inhibition.
Claims Coverage
The claims define a method encompassing exposing isolated lymphocytes to anthrax lethal toxin, contacting them with a test agent, and detecting lymphocyte-associated LT activity by measuring MAPKK-dependent cytokine responses or lymphocyte proliferation to identify agents that reduce anthrax pathogenicity.
Method for identifying agents decreasing anthrax pathogenicity via lymphocyte assays
A method comprising exposing isolated lymphocytes to anthrax lethal toxin (LT), contacting the lymphocytes with a test agent, and detecting lymphocyte-associated LT activity by either measuring a MAPKK-dependent cytokine response or lymphocyte proliferation; an increased cytokine response or increased proliferation in presence of the test agent indicates decreased LT activity and decreased anthrax pathogenicity.
Use of purified LT or Bacillus anthracis organisms for lymphocyte exposure
Exposing isolated lymphocytes to either purified anthrax LT or to an organism producing LT, such as Bacillus anthracis or its spores, to evaluate LT activity.
Stimulation of isolated lymphocytes post-exposure
Stimulating isolated lymphocytes after exposure to LT to induce MAPKK-dependent cytokine responses or proliferation, with stimulation methods including activating T-cell receptors by agents such as anti-CD3, anti-CD28, PMA plus ionomycin for T-cells, or activating B-cell receptors via agents like LPS, anti-CD40, or anti-IgM for B-cells.
Sequential contact with LT and test agent
Contacting lymphocytes first with LT, then with the test agent and stimulation, wherein decreased lymphocyte-associated LT activity indicates the test agent's therapeutic potential against anthrax.
Comparative and quantitative measurement of lymphocyte-associated LT activity
Comparing detected lymphocyte-associated LT activity to a baseline, control, or standard curve reflecting biological LT activity to assess the effect of test agents.
In vivo administration following in vitro agent identification
Selecting agents that reduce lymphocyte-associated LT activity in vitro, administering them to subjects, and determining if LT activity decreases in the subject, indicating reduced anthrax pathogenicity.
Application to human lymphocytes and specific cytokines
Performing the method with isolated human lymphocytes, including CD4+ T-cells and B-cells, detecting specific MAPKK-dependent cytokines such as IL-2, IL-4, IFN-γ, TNF-α, IL-1α, IL-1β, IL-6, IL-12, and IL-18, or measuring proliferation of stimulated lymphocytes.
In vitro assay execution
Conducting the method in vitro using isolated human lymphocyte cell lines such as Jurkat cells or primary cells isolated from human blood.
The claims cover methods to identify agents decreasing the pathogenicity of anthrax by measuring lymphocyte responses (cytokine production and proliferation) to anthrax LT exposure, with embodiments involving purified LT or Bacillus anthracis spores, use of stimulated lymphocytes, comparisons to controls or baselines, and application of identified agents in vivo to reduce anthrax LT activity.
Stated Advantages
The bioassays use human lymphocytes, providing a system more relevant to human anthrax pathology than previous murine macrophage-based assays.
The methods enable identification and evaluation of therapeutic agents that decrease anthrax LT activity by measuring biologically relevant human immune responses.
The assays permit quantitation of biologically active LT, distinguishing active versus neutralized toxin, aiding in diagnosis and therapeutic assessment.
The methodologies allow in vitro screening followed by in vivo confirmation, facilitating drug development against anthrax LT.
Documented Applications
Screening and identifying therapeutic agents that decrease the pathogenicity of anthrax by reducing lymphocyte-associated LT activity.
Determining the efficacy or potency of potential anti-anthrax therapeutic agents in vitro and in vivo.
Diagnosing anthrax infection in a subject by measuring lymphocyte-associated LT activity or biologically active anthrax LT levels.
Treating or preventing anthrax infection by administering agents that increase lymphocyte MAPKK-dependent cytokine production or proliferation, thereby mitigating LT effects.
Studying anthrax LT biological activity through in vitro bioassays using isolated lymphocytes from humans or cell lines.
Using in vivo models, such as non-human primates, rabbits, rats, or mice, to test candidate anthrax therapeutics identified in vitro.
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