# US 10,309,956 B2 · Process for assessing risk of substance administration > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,309,956 B2 - Document ID: US10309956B2 - Status: Granted - Kind: B2 - Application number: 14/796,091 - Filed: 2015-07-10 - Published: 2016-01-28 - Granted: 2019-06-04 - Earliest claimed: 2014-07-10 - Assignees: R.J. Reynolds Tobacco Company; Wake Forest University Health Sciences - Applicants: R. J. Reynolds Tobacco Company - Inventors: Gaddamanugu L. Prasad; Subhashini Arimilli - Web page: https://patentgrep.com/patent/US10309956B2 ## CPC - G01N 33/5023: {on expression patterns} - G01N 33/5047: {Cells of the immune system} - G01N 2800/50: Determining the risk of developing a disease - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract A process for assessing risk that administration of a substance will suppress immune competence in a population of subjects is provided. Such a substance may be a substance related to, contained in or derived from a tobacco product or, more generally, a substance concerning which it is desired to assess risk of administration of the substance. ## Claims ### Claim 1 A process for assessing risk that administration of a tobacco product preparation (TPP) substance derived from tobacco will suppress immune competence in a population of subjects, the process comprising: collecting a sample of blood from each a plurality of subjects, said subjects being members of the population; isolating peripheral blood mononuclear cells (PBMCs) from the sample of blood to form isolated PBMCs; administering the TPP substance to the isolated PBMCs for 3 hours, thereby forming substance-exposed PBMCs; stimulating the substance-exposed PBMCs with a Toll-like receptor ligand to form Toll-like receptor-ligand stimulated substance-exposed PBMCs, wherein the stimulating takes place for a duration of less than 6 hours; measuring one or more levels of a cytokine in the Toll-like receptor-ligand stimulated substance-exposed PBMCs to form one or more measured responses; aggregating the one or more measured responses from each of the plurality of subjects to form one or more response aggregates; comparing the one or more response aggregates to one or more immune-competence-associated reference ranges measured in control cells, thereby assessing risk that administration of the substance will suppress immune competence in the population of subjects. ### Claim 2 (depends on 1) The process according to claim 1, wherein the substance derived from tobacco comprises total particulate matter. ### Claim 3 (depends on 1) The process according to claim 1, wherein the tobacco product is a smokeless tobacco product. ### Claim 4 (depends on 1) The process according to claim 1, wherein the stimulating takes place for a duration of no more than 5 hours. ### Claim 5 (depends on 1) The process according to claim 1, wherein the cytokine is TNF-α. ### Claim 6 (depends on 1) The process according to claim 1, wherein the cytokine is IFN-γ. ### Claim 7 (depends on 1) The process according to claim 1, wherein the cytokine is IL-1β. ### Claim 8 (depends on 1) The process according to claim 1, wherein the cytokine is IL-10. ### Claim 9 (depends on 1) The process according to claim 1, wherein the cytokine is IL-12. ### Claim 10 (depends on 1) The process according to claim 1, wherein the Toll-like receptor ligand comprises a bacterial lipopolysaccharide. ### Claim 11 (depends on 1) The process according to claim 1, wherein the Toll-like receptor ligand comprises polyinosinic:polycytidylic acid (Poly I:C). ### Claim 12 (depends on 1) The process according to claim 1, additionally comprising administering phorbol 12-myristate 13-acetate (PMA) and Ionomycin (Iono) (PMA/Iono) to the substance-exposed PBMCs. ### Claim 13 A process for assessing risk that administration of a tobacco product preparation (TPP) substance derived from tobacco will suppress immune competence in a population of subjects, the process comprising: collecting a sample of blood from each a plurality of subjects, said subjects being members of the population; isolating peripheral blood mononuclear cells (PBMCs) from the sample of blood to form isolated PBMCs; administering the TPP substance derived from tobacco to the isolated PBMCs for 3 hours, thereby forming substance-exposed PBMCs; stimulating the substance-exposed PBMCs with phorbol 12-myristate 13-acetate (PMA) and Ionomycin (Iono) to form PMA/Iono stimulated substance-exposed PBMCs, wherein the stimulating takes place for a duration of less than 6 hours; measuring one or more responses of levels of a cytokine in the PMA/Iono stimulated substance-exposed PBMCs to form one or more measured responses; aggregating the one or more measured responses from each of the plurality of subjects to form one or more response aggregates; comparing the one or more response aggregates to one or more immune-competence-associated reference ranges measured in control cells, thereby assessing risk that administration of the substance will suppress immune competence in the population of subjects. ### Claim 14 A process for assessing risk that administration of a tobacco product preparation (TPP) substance derived from tobacco will suppress immune competence in a population of human subjects, the process comprising: collecting a sample of blood from each a plurality of human subjects, said subjects being members of the population; isolating peripheral blood mononuclear cells (PBMCs) from the sample of blood to form isolated PBMCs; administering the TPP substance to the isolated PBMCs for 3 hours, thereby forming substance-exposed PBMCs; stimulating the substance-exposed PBMCs with a Toll-like receptor ligand to form Toll-like receptor-ligand stimulated substance-exposed PBMCs, wherein the stimulating takes place for a duration of less than 6 hours; measuring one or more levels of a cytokine in the Toll-like receptor-ligand stimulated substance-exposed PBMCs to form one or more measured responses; aggregating the one or more measured responses from each of the plurality of subjects to form one or more response aggregates; comparing the one or more response aggregates to one or more immune-competence-associated reference ranges measured in control cells, thereby assessing risk that administration of the substance will suppress immune competence in the population of human subjects. ### Claim 15 A process for assessing risk that administration of a tobacco product preparation (TPP) substance derived from tobacco will suppress immune competence in a population of human subjects, the process comprising: collecting a sample of blood from each a plurality of human subjects, said subjects being members of the population; isolating peripheral blood mononuclear cells (PBMCs) from the sample of blood to form isolated PBMCs; administering the TPP substance to the isolated PBMCs for 3 hours, thereby forming substance-exposed PBMCs; stimulating the substance-exposed PBMCs with PMA/Iono stimulated substance-exposed PBMCs, wherein the stimulating takes place for a duration of less than 6 hours; measuring one or more levels of a cytokine in the PMA/Iono stimulated substance-exposed PBMCs to form one or more measured responses; aggregating the one or more measured responses from each of the plurality of subjects to form one or more response aggregates; comparing the one or more response aggregates to one or more immune-competence-associated reference ranges measured in control cells, thereby assessing risk that administration of the substance will suppress immune competence in the population of human subjects. ### Claim 16 A process for assessing risk that administration of a tobacco product preparation (TPP) substance derived from tobacco will suppress immune competence in a population of subjects, the process comprising: collecting a sample of blood from each a plurality of subjects, said subjects being members of the population; isolating peripheral blood mononuclear cells (PBMCs) from the sample of blood to form isolated PBMCs; cryopreserving the isolated PBMCs at temperature less than zero degrees Celsius; warming the isolated PBMCs to a temperature higher than zero degrees Celsius; administering the TPP substance to the isolated PBMCs for 3 hours, thereby forming substance-exposed PBMCs; stimulating the substance-exposed PBMCs with a Toll-like receptor ligand to form Toll-like receptor-ligand stimulated substance-exposed PBMCs, wherein the stimulating takes place for a duration of less than 6 hours; measuring one or more levels of a cytokine in the Toll-like receptor-ligand stimulated substance-exposed PBMCs to form one or more measured responses; aggregating the one or more measured responses from each of the plurality of subjects to form one or more response aggregates; comparing the one or more response aggregates to one or more immune-competence-associated reference ranges measured in control cells, thereby assessing risk that administration of the substance will suppress immune competence in the population of subjects. ### Claim 17 A process for assessing risk that administration of a tobacco product preparation (TPP) substance derived from tobacco will suppress immune competence in a population of subjects, the process comprising: collecting a sample of blood from each a plurality of subjects, said subjects being members of the population; isolating peripheral blood mononuclear cells (PBMCs) from the sample of blood to form isolated PBMCs; cryopreserving the isolated PBMCs at temperature −150 degrees Celsius; warming the isolated PBMCs to a temperature 37 degrees Celsius; administering the TPP substance to the isolated PBMCs for 3 hours, thereby forming substance-exposed PBMCs; stimulating the substance-exposed PBMCs with PMA/Iono to form stimulated substance-exposed PBMCs, wherein the stimulating takes place for a duration of less than 6 hours; measuring one or more levels of a cytokine in the PMA/Iono stimulated substance-exposed PBMCs to form one or more measured responses; aggregating the one or more measured responses from each of the plurality of subjects to form one or more response aggregates; comparing the one or more response aggregates to one or more immune-competence-associated reference ranges measured in control cells, thereby assessing risk that administration of the substance will suppress immune competence in the population of subjects.