US 5,514,555 A · Assays and therapeutic methods based on lymphocyte chemoattractants

Abstract

The present invention relates to a novel assay for lymphocyte chemotaxis. The assay is transendothelial assay using endothelial cells cultured on microporous filters. Lymphocyte transmigration through the filter toward a suspected chemoattractant is measured. Apparatuses for carrying out the assay are also provided. The apparatuses and methods of the present invention can be used for the identification of inhibitors (e.g., antagonists) or promoters (chemoattractants) of the adhesion receptor-mediated migration of leukocytes through the endothelium (extravasation). Such inhibitors and promoters respectively inhibit and promote the inflammatory response, and thus have therapeutic utilities. The inhibitors and promoters are identified by detecting their abilities to respectively inhibit or promote the chemotaxis of lymphocytes in the assay of the invention. The assay of the invention also has diagnostic utilities for detecting a disease or disorder involving a defect in lymphocyte chemotaxis. In a specific embodiment, the invention provides a novel lymphocyte chemoattractant, termed LCA, of molecular weight of about 14,500.+-.3,000 daltons. Derivatives and analogs of LCA, and antibodies and antibody fragments thereto are also provided. The invention also relates to therapeutic uses and compositions related to the foregoing.

Claims23

Dependent claims

A method for detecting or measuring lymphocyte chemotaxis comprising detecting or measuring the transmigration of lymphocytes completely through a filter in a direction (a) toward increased levels of a known or suspected lymphocyte chemoattractant, and (b) from a first surface of the filter toward an opposite, second surface of the filter;

wherein said filter is a microporous filter having on its first surface an endothelial cell monolayer;
and wherein the presence and amount of said transmigration of lymphocytes completely through the filter indicates the presence and amount, respectively, of lymphocyte chemotaxis.

A method for detecting or measuring lymphocyte chemotaxis comprising:

(a) incubating an apparatus for a time period sufficient to allow any chemotaxis of lymphocytes to occur, in which said apparatus comprises:
(i) a microporous filter;
(ii) an endothelial cell monolayer on an upper surface of said filter;
(iii) a first chamber having an opening communicating with said endothelial cell monolayer, said first chamber containing a first composition, said first composition comprising a first fluid and lymphocytes;
in which said first fluid contacts said endothelial cell monolayer;
and
(iv) a second chamber having an opening communicating with a lower surface of said filter;
in which said second chamber contains a second composition;
said second composition (A) comprising a second fluid contacting the lower surface of said filter, and (B) comprising or suspected of comprising molecules having lymphocyte chemoattractive activity;
and
(b) measuring the amount of lymphocytes in the second chamber, in which an increased amount of lymphocytes in the second chamber relative to the baseline amount present when said second composition does not comprise a lymphocyte chemoattractant indicates that lymphocyte chemotaxis has occurred, and in which the difference between said increased amount and the baseline amount indicates the amount of lymphocyte chemotaxis.

8 more elements

Dependencies are inferred from the claim text.

Assays and therapeutic methods based on lymphocyte chemoattractants

Life of this patent

Assays and therapeutic methods based on lymphocyte chemoattractants

Life of this patent

Abstract

Claims23