Internet for Pharmaceutical and Biotech Communities
| Newsletter | Advertising |
 
 
 

  

Pharm/Biotech
Resources

Outsourcing Guide

Cont. Education

Software/Reports

Training Courses

Web Seminars

Jobs

Buyer's Guide

Home Page

Pharm Patents /
Licensing

Pharm News

Federal Register

Pharm Stocks

FDA Links

FDA Warning Letters

FDA Doc/cGMP

Pharm/Biotech Events

Consultants

Advertiser Info

Newsletter Subscription

Web Links

Suggestions

Site Map
 

 
   



 

Title:  Method to identify regulators of cellular activation using Bcl10
United States Patent: 
7,169,570
Issued: 
January 30, 2007

Inventors: 
Schaefer; Brian C. (Columbia, MD), Marrack; Philippa (Denver, CO), Kappler; John W. (Denver, CO)
Assignee: 
National Jewish Medical and Research Center (Denver, CO)
Appl. No.: 
10/795,157
Filed: 
March 4, 2004


 

Woodbury College's Master of Science in Law


Abstract

Disclosed are methods for evaluating the activation of Bcl10 in a cell in response to a putative stimulus, as well as methods for evaluating or identifying a regulatory compound which regulates activation of Bcl10-mediated signal transduction. These methods utilize the discovery of the activation-dependent formation in a cell of Bcl10 aggregates in a cell.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a method for evaluating activation of Bcl10 in a cell in response to a putative stimulus. The method includes the steps of: (a) contacting an isolated cell having a Bcl10 signal transduction pathway with a putative stimulus; and (b) detecting whether Bcl10 expressed by the cell polymerizes into aggregates in the cell after contact with the putative stimulus as compared to in the absence of the putative stimulus. The polymerization of Bcl10 into aggregates in the cell indicates Bcl10-mediated activation of the cell.

The step of detecting can be performed by any suitable detection method, including, but not limited to, using microscopy to visualize Bcl10 in the cell (e.g., using fluorescent microscopy to visualize Bcl10 in the cell); using an antibody that selectively binds to Bcl10; detecting a recombinant Bcl10 protein expressed by the cell; detecting a Bcl10-reporter fusion protein expressed by the cell; using biochemical extraction of Bcl10 from cells and detection of Bcl10 aggregates by biochemical fractionation techniques (e.g., gel electrophoresis and gradient centrifugation); measuring changes in a refractive index of intact cells or biochemical extracts of cells; and measuring changes in the light scatter properties of cells.

In one embodiment, the method further includes a step of detecting translocation of the aggregates of Bcl10 to a site of contact between the cell and the putative stimulus, the translocation being further indicative of Bcl10-mediated activation of the cell. In another embodiment, the method further includes a step of detecting whether there is change in the level of Bcl10 expression in the cell in the presence of the putative stimulus as compared to the level of Bcl10 expression in the absence of the putative stimulus, a change in Bcl10 expression being further indicative Bcl10-mediated activation of the cell. In another embodiment, the method further includes a step of detecting whether Bcl10 is phosphorylated in the cell, the phosphorylation of Bcl10 being further indicative of Bcl10-mediated activation of the cell.

In one embodiment, the cell is a lymphocyte expressing an antigen receptor, and the polymerization of Bcl10 into aggregates in the cell in the presence of the putative stimulus indicates activation of the cell through an antigen receptor-associated, Bcl10 signal transduction pathway.

For example, in one aspect of the invention, the cell is a T lymphocyte, which can include, but is not limited to, a primary lymph node T lymphocyte, a primary splenic T lymphocyte, a T lymphocyte from a transgenic mouse, a T lymphocyte clone or a T lymphocyte hybridoma. In this aspect of the invention, the method can include the following steps: (a) culturing a T lymphocyte expressing a T cell receptor (TCR) with a putative stimulus for activating the T lymphocyte through the TCR signal transduction pathway; and (b) detecting whether Bcl10 expressed by the T lymphocyte polymerizes into aggregates in the T lymphocyte in the presence of the putative stimulus as compared to in the absence of the putative stimulus, wherein the polymerization of Bcl10 into aggregates in the T lymphocyte indicates Bcl10-mediated activation of the T lymphocyte.

In this aspect of the invention, the putative stimulus can include, but is not limited to, an antigen presenting cell expressing a major histocompatibility complex (MHC)-antigen complex wherein the antigen is bound to an antigen binding site of the MHC; an antibody that selectively binds to and activates the TCR; an antibody that selectively binds to and activates CD3; a purified, soluble MHC-peptide complex; a T lymphocyte mitogen (e.g., PHA, conconavalin A, a phorbol ester); an activator of protein kinase C (PKC); and a T lymphocyte superantigen. In one aspect, the step of culturing comprises culturing the T lymphocyte with an antigen presenting cell expressing an MHC-antigen complex, wherein the antigen is bound to an antigen binding site of the MHC, wherein, if the TCR binds to the MHC-antigen complex, an antigen-specific site of contact between the T lymphocyte and the antigen presenting cell is formed.

In another aspect of this embodiment of the invention, the lymphocyte is a B lymphocyte, which can include, but is not limited to, a primary B lymphocyte, a B lymphocyte from a transgenic mouse, or a B lymphocyte hybridoma. In this aspect of the invention, the method can include the steps of: (a) culturing a B lymphocyte expressing a B cell antigen receptor (BCR) with a putative stimulus for activating the B lymphocyte through the BCR; and (b) detecting whether Bcl10 expressed by the B lymphocyte polymerizes into aggregates in the B lymphocyte in the presence of the putative stimulus as compared to in the absence of the putative stimulus, wherein the polymerization of Bcl10 into aggregates in the B lymphocyte indicates Bcl10-mediated activation of the lymphocyte.

In this aspect of the invention, the putative stimulus can include, but is not limited to, an antibody that selectively binds to and activates the BCR; an activator of protein kinase C (PKC); a phorbol ester; antibodies that selectively bind to and activate transmembrane forms of IgM, IgD, IgG, IgA or IgE; antibodies that selectively bind to and activate the immunoglobulin-associated signaling molecules Ig-.alpha. or Ig-.beta.; and polyvalent ligands for IgM, IgD, IgG, IgA or IgE (e.g., polyvalent cognant antigen, lectins that bind immunoglobulin, and compounds that aggregate surface immunoglobulin).

Yet another embodiment of the present invention relates to a method to identify a regulatory compound which regulates activation of Bcl10-mediated signal transduction. The method includes the steps of: (a) contacting an isolated cell having a Bcl10 signal transduction pathway with a putative regulatory compound and a stimulus, under conditions in which, in the absence of the putative regulatory compound, the stimulus activates the Bcl10 signal transduction pathway such that Bcl10 expressed by the cell polymerizes into aggregates in the cell; and (b) detecting whether the putative regulatory compound increases or decreases the level of the aggregates of Bcl10 in the cell in as compared to in the absence of the putative regulatory compound, wherein an increase or decrease in the level of the aggregates of Bcl10 in the presence of the putative regulatory compound indicates that the compound regulates Bcl10-mediated signal transduction in the cell. A decrease in the level of the aggregates of Bcl10 in the presence of the putative regulatory compound indicates that the compound decreases Bcl10 signal transduction. An increase in the level of the aggregates of Bcl10 in the presence of the putative regulatory compound indicates that the compound increases Bcl10 signal transduction.

In one aspect, the step of detecting includes detecting whether the putative regulatory compound inhibits the translocation of the aggregates of Bcl10 to the site of contact between the cell and the stimulus, wherein such inhibition indicates that the putative regulatory compound is an inhibitor of Bcl10 signal transduction. The step of detecting can also include, but is not limited to, using microscopy to visualize Bcl10 in the cell (e.g., using fluorescent microscopy to visualize Bcl10 in the cell), detecting Bcl10 using an antibody that selectively binds to Bcl10, detecting a recombinant Bcl10 protein expressed by the cell, detecting a Bcl10-reporter fusion protein expressed by the cell, using biochemical extraction of Bcl10 from cells and detection of Bcl10 aggregates by biochemical fractionation techniques, measuring changes in a refractive index of intact cells or biochemical extracts of cells, and measuring changes in the light scatter properties of cells. In one embodiment, the method is performed as a high-throughput assay for screening multiple putative regulatory compounds simultaneously.

In one aspect of this embodiment of the invention, the step of contacting the cell with a putative regulatory compound is performed before the step of culturing the cell with the stimulus. In another aspect, the method further includes a step of detecting whether the putative regulatory compound increases or decreases the translocation of the aggregates of Bcl10 to a site of contact between the cell and the putative stimulus, wherein an increase in translocation indicates an increase in Bcl10 signal transduction and wherein a decrease in translocation indicates a decrease in Bcl10 signal transduction. In another aspect, the method further includes a step of detecting a change in a characteristic of Bcl10 activity selected from the group consisting of a change in the level of Bcl10 expression and a change in the level of phosphorylation of Bcl10 in the cell, wherein a change in the level of Bcl10 expression or a change in the level of Bcl10 phosphorylation in the presence of the putative regulatory compound indicates that the putative regulatory compound regulates Bcl10 signal transduction.

In one aspect of this embodiment of the invention, the cell is a T lymphocyte. For example, the method can include the steps of: (a) culturing a T lymphocyte having a T cell antigen receptor (TCR) with a stimulus that activates the T lymphocyte through the TCR signal transduction pathway; (b) contacting the T lymphocyte with a putative regulatory compound under conditions in which, in the absence of the putative regulatory compound, Bcl10 polymerizes by forming aggregates in the T lymphocyte; and (c) detecting whether the level of the aggregates of Bcl10 in the T lymphocyte increases or decreases in the presence of the putative regulatory compound as compared to in the absence of the putative regulatory compound, wherein an increase or decrease in the level of the aggregates of Bcl10 in the presence of the putative regulatory compound indicates that the compound regulates the Bcl10-mediated signal transduction in the cell.

In another aspect of this embodiment of the invention, the cell is a B lymphocyte. For example, the method can include the steps of: (a) culturing a B lymphocyte having a B cell antigen receptor (BCR) with a stimulus that activates the B lymphocyte through the BCR signal transduction pathway; (b) contacting the B lymphocyte with a putative regulatory compound under conditions in which, in the absence of the putative regulatory compound, Bcl10 polymerizes by forming aggregates in the B lymphocyte; and (c) detecting whether the level of the aggregates of Bcl10 in the B lymphocyte increases or decreases in the presence of the putative regulatory compound as compared to in the absence of the putative regulatory compound, wherein an increase or decrease in the level of the aggregates of Bcl10 in the presence of the putative regulatory compound indicates that the compound regulates the Bcl10-mediated signal transduction in the cell.
 


Claim 1 of 47 Claims

1. A method for evaluating activation of Bcl10 in a cell in response to a putative stimulus: a) contacting an isolated cell having a Bcl10 signal transduction pathway with a putative stimulus; and b) detecting whether Bcl10 expressed by the cell polymerizes into punctate and filamentous structures in the cell after contact with the putative stimulus as compared to in the absence of the putative stimulus, wherein the polymerization of Bcl10 into punctate and filamentous structures in the cell indicates Bcl10-mediated activation of the cell.

 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
[ Outsourcing Guide ] [ Cont. Education ] [ Software/Reports ] [ Training Courses ]
[ Web Seminars ] [ Jobs ] [ Consultants ] [ Buyer's Guide ] [ Advertiser Info ]

[ Home ] [ Pharm Patents / Licensing ] [ Pharm News ] [ Federal Register ]
[ Pharm Stocks ] [ FDA Links ] [ FDA Warning Letters ] [ FDA Doc/cGMP ]
[ Pharm/Biotech Events ] [ Newsletter Subscription ] [ Web Links ] [ Suggestions ]
[ Site Map ]