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WO2011064351A1 - Récepteur de quinine b1 comme biomarqueur d'une inflammation des voies aériennes liée à une exposition à la fumée - Google Patents

Récepteur de quinine b1 comme biomarqueur d'une inflammation des voies aériennes liée à une exposition à la fumée Download PDF

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Publication number
WO2011064351A1
WO2011064351A1 PCT/EP2010/068348 EP2010068348W WO2011064351A1 WO 2011064351 A1 WO2011064351 A1 WO 2011064351A1 EP 2010068348 W EP2010068348 W EP 2010068348W WO 2011064351 A1 WO2011064351 A1 WO 2011064351A1
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Prior art keywords
bir
cell
smoke
expression level
lung
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Inventor
Rejean Couture
James Lin
Andre Morin
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Universite de Montreal
Imperial Tobacco Canada Ltd
British American Tobacco Investments Ltd IFI
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Universite de Montreal
Imperial Tobacco Canada Ltd
British American Tobacco Investments Ltd IFI
British American Tobacco Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2410/00—Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids
    • G01N2410/06—Kallidins; Bradykinins; Related peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/12—Pulmonary diseases

Definitions

  • the disclosure relates to methods for detecting airway inflammation, such as mouth, trachea or lung inflammation, by measuring biomarker upregulation.
  • the disclosure relates to detecting airway inflammation caused by smoke, such as tobacco smoke.
  • kinins are peptides that cause certain pain and inflammation responses. They activate two G-protein coupled receptors (GPCR) named bradykinin B 2 receptor (B 2 R) and bradykinin Bi receptor (BiR). B 2 R is constitutively expressed at a relatively constant level. BiR is inducible as a response to certain substances, but the response, if any, varies depending on the type of compound and the type of tissue affected. Tissue insult, bacterial toxins and oxidative stress are well known inducers of BiR. There are numerous other compounds that are involved in inflammation. The biochemical changes are not only in the inflamed cell but may involve immune cells recruited to the site of the inflammation. In this complex environment, it can be difficult to determine whether a particular compound causes, exacerbates or reduces inflammation.
  • GPCR G-protein coupled receptors
  • bradykinin BK
  • kallidin Lys-BK
  • pro-inflammatory vasoactive kinin peptides (9-11 amino-acids). They are present in inactive form in blood and tissues. Tissue injury triggers the formation of active kinins, which then causes vasodilatation, vascular hyperpermeability, leukocytes infiltration, pain and hyperalgesia.
  • mediators prostaglandins, leukotriens, nitric oxide, cytokines, histamine and serotonin
  • TRPV1 transient receptor potential vanilloid 1
  • Kinins act through these different mechanisms to facilitate the release of substances from primary sensory nerve terminals (substance P, calcitonin gene related peptide, nitric oxide, glutamate) thereby causing pain and neurogenic inflammation (Couture et al., 2001 ; Planells-Cases et al., 2005; Singh Tahim et al., 2005).
  • B-iR is a useful biomarker for inflammation in trachea, lung and mouth cells, for example, inflammation related to smoke exposure.
  • the invention shows 1) BiR induction (mRNA and protein) by exposing ex vivo tissue slices and cultured cells to cigarette smoke condensate (CSC), 2) the cellular localisation of the induced BiR, and 3) the mechanism of BiR induction after CSC treatment.
  • CSC cigarette smoke condensate
  • the invention relates to a method of determining whether a test substance causes airway cell inflammation comprising
  • B-iR bradykinin B1 receptor
  • B-iR level Prior to contact with the test substance, B-iR level may be not expressed at all in the sample or it maybe expressed at a low level that is close to, or below, the limit of detection.
  • DMSO dimethyl sulfoxide
  • CSC smoke condensates
  • FIG. 2A illustrates the time-course (2h, 4h, 8h, 24h) effect of CSC on BiR, IL-1 ⁇ and TNF-a mRNA expression in A549.
  • Cells were either left untreated or treated with IL-1 ⁇ (positive control, 50 pg/ml; 24h), DMSO (vehicle, 0.13%) or 10 pg/ml of CSC for designated hours before ribonucleic acid (RNA) extraction.
  • RNA ribonucleic acid
  • Figure 3A illustrates the induction of BiR, B 2 R, IL-1 ⁇ and TNF-a mRNA in rat lung slices after 24h treatment with: DMSO (vehicle; 0.13%), IL-1 ⁇ (positive control; 50 pg/ml) and 5 concentrations of smoke condensates (5, 10, 12.5, 20 and 30 pg/ml).
  • DMSO vehicle
  • IL-1 ⁇ positive control
  • 5 concentrations of smoke condensates 5, 10, 12.5, 20 and 30 pg/ml
  • FIG. 4A illustrates the time-course effect of CSC treatment on BiR, IL-1 ⁇ and TNF-a mRNA expression in rat lung slices.
  • Lung slices were either left untreated or treated with 5 pg/ml CSC for various durations (2h, 4h, 8h, 24h) before they were harvested for RNA extraction.
  • DMSO 0.13% was given to lung slices for the same durations.
  • IL-1 ⁇ 50 pg/ml
  • Fold of change of each time point was obtained by normalising CSC treated group to the DMSO group of the same treatment duration.
  • FIG 5 illustrates the measurement of mRNA expression of BiR and IL-1 ⁇ in LS by quantitative real-time PCR.
  • Figure 7 illustrates the expression of BiR and B 2 R by qPCR in the spinal cord of streptozotocine (STZ)-treated diabetic male rats. These results were obtained by method #2. Specific data are contained in Table 4.
  • Figure 8 illustrates the expression of B ⁇ R and B 2 R by qPCR in male rat lung cells. These results were obtained by method #1. Specific data are contained in Table 5.
  • Figure 9 illustrates the expression of BiR and B 2 R by qPCR in paraffin sections of male rat trachea isolated from samples B, C and D when compared to A (control). Samples B, C and D represent CSC from different types of tobacco. These results were obtained by method #1. Specific data are contained in Table 6.
  • Figure 10 illustrates the expression of B-
  • Figure 11 illustrates the expression of BiR and B 2 R by qPCR in fixed male rat trachea isolated from B, C and D when compared to A (control). These results were obtained by method #2. Specific data are contained in Table 8.
  • Figure 12 illustrates the expression of BiR and B 2 R by qPCR in female rat trachea isolated from B, C and D when compared to A (control). These results were obtained by method #2. Specific data are contained in Table 9.
  • Figure 13 illustrates the induction of B ⁇ (A), IL-1 ⁇ (B) and TNF-a (C) genes in rat lung slices exposed to whole smoke (WS) or vapor phase (VP) for 30 min at a dilution of 2% smoke in air.
  • Lung slices were harvested for gene expression analysis using real-time PCR following a 1 day treatment and a 24h recovery. *lndicates statistical difference at p ⁇ 0.01 and indicates a difference at p ⁇ 0.05. For each treatment, results were obtained from two independent experiments with at least four different rats each (n> 4).
  • the airway is the portion of the respiratory system through which air flows when traveling from the atmosphere to the alveoli. It includes the mouth, trachea and lungs.
  • the invention establishes that B-
  • the airway includes indigenous cells, such as epithelial cells, and also infiltrating cells, such as neutrophils of the immune system.
  • airway cells optionally include both cells indigenous to the airway and cells that have infiltrated the airway.
  • the invention further provides the first identification of BiR as a biomarker for tobacco smoke-mediated inflammation.
  • This application provides methods of using B-iR to detect inflammation in airway cells. These methods are useful to determine the amount of B-iR upregulation and inflammation caused by different types of smoke. BiR upregulation and inflammation caused by other types of test substances is also readily determined.
  • the methods of determining whether a test substance causes airway cell inflammation optionally include
  • the determining step is typically done by reference to the E ⁇ R expression level in the cell prior to contact with the test substance.
  • R in the cell is determined (also called a background level).
  • the determining step comprises comparing the expression level after the contacting step (post-contact expression level) to the pre-contact expression level of BiR and determining whether the expression level of B-
  • the determining step is done by reference to a control (e.g. control sample or control population) or a pre-determined standard result.
  • a control e.g. control sample or control population
  • a pre-determined standard result For example, a higher expression level of BiR compared to either a negative control or to a pre-determined, inflamed standard result indicates that the substance causes inflammation. Determining the occurrence of comparable, or lower, expression in the test sample in such a comparison would indicate that no inflammation occurred. An expression level of B-
  • the methods are useful to compare the effects of different types of smoke on B-iR. For example, different strains of tobacco plants, or mixtures thereof, are readily tested in the assay. The effects of different components of delivery devices, such as filters on a particular type of smoke can also be examined. For example, filters having different sizes and arrangements of perforations to dilute smoke with air may be compared to assess BiR expression level using the assay.
  • a substance that reduces BiR expression level is an antiinflammatory agent.
  • a compound is administered together with, or separately from, the smoke to determine if the compound reduces B-
  • a potential anti-inflammatory agent may be administered to the sample before, concurrently with, or after the smoke.
  • kits for performing the methods optionally include at least one, two or three of the following: i) lung, trachea or mouth cells, and ii) an agent for determining BiR expression level, such as a) oligonucleotide primers that hybridize to B-
  • the kits are useful for assaying a test substance, such as a plant substance to be burned to produce smoke (e.g. tobacco).
  • the agents that detect the expression level are labeled with a detectable marker. Detection can be direct or indirect.
  • the detection agent in the kit can be for detecting a protein of interest (e.g. antibodies) or can be for detecting nucleic acid expression products encoding the proteins of interest (e.g. probes or primers).
  • the trachea, lung and mouth represent sensitive organs that provide tissue and cells for evaluation of the influence of inhaled cigarette smoke on gene expression.
  • An organ is a structure that contains at least two different types of tissue functioning together for a common purpose.
  • Trachea a main portion of the airway that runs from the base of the throat down to the lungs, is a major organ of the respiratory system.
  • Mouth which includes the upper opening of the digestive tract, beginning with the lips and including the gums, and tongue, is considered one of the major organs of the digestive system.
  • the sample is typically a cell culture, a tissue, an organ or a portion of a tissue or organ.
  • the portion may be a slice or other section.
  • cells are dissociated from the tissue or organ.
  • the test substance is optionally a gas or an aerosol mixture of gases and particles, such as smoke, which is typically used for experiments with subjects, such as mammals (e.g. rat, mouse or human cells or tissues).
  • the test substance is a liquid (e.g. liquid having particles suspended therein), such as smoke condensate or extract (e.g. cigarette smoke condensate or cigarette smoke extract), which is primarily used for experiments in vitro or ex vivo.
  • the smoke is typically from a plant substance used for smoking, such as tobacco.
  • interleukin-1 ⁇ is administered to the cell in addition to the test substance, typically when testing a cell culture.
  • the inventors' results in lung cells show that B-
  • R is a useful biomarker of trachea inflammation related to cigarette smoke exposure.
  • experiments on trachea showed that BiR is induced and overexpressed in mammalian trachea exposed to inhaled cigarette smoke for a period of six weeks (quantitative RT-PCR).
  • Cigarette smokes of different composition had distinctive effect on B-iR expression.
  • the sample C treatment caused a greater induction than A, B and DCR.
  • the expression of B 2 R was not affected as significantly as the BiR by inhaled cigarette smoke in fixed trachea.
  • B-iR will also be upregulated in buccal epithelial cells.
  • BiR is a useful biomarker of buccal epithelial cell inflammation related to tobacco smoke exposure, for example exposure to smoke from cigarettes.
  • the inventors conduct experiments on buccal epithelial cells to show that B-iR is induced and overexpressed in mammalian buccal epithelial cells exposed to inhaled cigarette smoke (quantitative RT-PCR).
  • Buccal epithelial cells come in contact with whole smoke, similar to epithelial cells coating the trachea and the lung. More chemicals from the smoke may contact the ceils from the mouth than the tracheal and lung cells. Due to deposition, selective filtration or adsorption and condensation, less smoke chemicals will reach the trachea and the lungs during inhalation, in comparison to the mouth.
  • the tobacco smoke condensate comprises smoke condensate (such as CSC)
  • the smoke is typically delivered to the cell culture in a dose of 0.1 Mg/ml, 0.25 pg/ml or
  • BiR RNA expression in the cell culture is typically upregulated at least: 5-fold, 10-fold or 20-fold.
  • the IL-1 ⁇ mRNA expression in the cell culture is typically upregulated at least: 5-fold or 10-fold.
  • the expression level of BiR RNA is then detected in the cell after the contacting step. For example, B-
  • R protein is optionally detected in the tissue 2-12 or 4-9 hours after the contacting step, optionally 7.5-8.5 hours after the contacting step. Alternatively, or additionally, the expression level of B-
  • the B1 R RNA expression in the tissue is typically upregulated at least: 5-fold, 10-fold or 20- fold.
  • the B1 R protein expression level in the tissue is typically upregulated at least: 1.5, 2 or 3-fold.
  • the smoke condensate test substance is typically delivered to the sample in a dose comprising 0.1 g ml or 3 pg/ml to 8 pg/ml, 10 pg/ml. Similar dosages may be used for smoke extract. Smoke may also be applied to tissues. Where live animals are used for tests, the animal is typically exposed to smoke for at least:
  • the B ⁇ RNA expression level in tissue is measured 0.5-5 or 1-3 hours after the contacting step, optionally 1.5-2.5 hours.
  • the sample optionally comprises lung, trachea or mouth tissue and the BiR RNA expression level is typically measured 20-28 or 23-25 hours after the contacting step, optionally 23.5-24.5 hours.
  • the sample optionally comprises lung, trachea or mouth tissue and the B ⁇ R protein expression level is measured 5-12 or 7-9 hours after the contacting step, optionally 7.5-8.5 hours.
  • the B1 R RNA expression in the tissue is typically upregulated at least: 5-fold, 10-fold or 20- fold.
  • the B1 R protein expression level in the tissue is typically upregulated at least: 1.5, 2 or 3-fold.
  • the determining step is optionally qualitative or quantitative.
  • Various methods are known in the art for detecting RNA and protein levels.
  • the amount of a protein is determined, for example, by immunoassays such as Western blots, ELISA or immunoprecipitation followed by SDS-PAGE immunocytochemistry.
  • protein arrays including microarrays, can be used.
  • Protein based protocols typically use agents that bind with specific affinity to the protein of interest.
  • the agents are antibodies or antibody fragments that can be readily generated using techniques known in the art. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals.
  • antibody includes antibody fragments, such as Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof and bispecific antibody fragments, all of the foregoing being readily generated using known methods.
  • F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab 1 and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.
  • Other affinity binding agents, such as aptamers, are optionally used in the methods.
  • Antibodies having specificity for BiR are prepared by conventional methods.
  • a mammal e.g. a mouse, hamster, or rabbit
  • an immunogenic form of the peptide which elicits an antibody response in the mammal.
  • Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art.
  • the peptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
  • antibody producing cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.
  • the agents such as antibodies or antibody fragments, that bind to BiR are labeled with a detectable marker.
  • the label is preferably capable of producing, either directly or indirectly, a detectable signal.
  • the label may be radio-opaque or a radioisotope, such as 3 H, 14 C, 32 P, 35 S, 123 l, 125 l or 31 1; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.
  • RNA transcripts within a sample, including hybridization or amplification assays.
  • assays include arrays (including microarrays), RT-PCR (including quantitative RT-PCR), nuclease protection assays and northern blots.
  • detection agents include probes and primers specific for the protein of interest.
  • Inhibition agents that reduce BiR [0051] The present invention also includes the identification of substances to reduce BiR expression. Substances that antagonize B-
  • Substrates that affect B ⁇ expression level are readily identified by exposing a sample described herein to smoke and a test inhibition agent and then assaying for BiR expression level.
  • R expression level optionally comprise:
  • R bradykinin B1 receptor
  • Examples of compounds identified using such methods include IL- 1 R antagonist, SN-50 and NAC, which showed strong inhibition of B ⁇ expression. Pentox was identified as having a partial inhibitory effect on CSC- induced BiR expression using the methods.
  • BiR mRNA is maximally induced after 4h exposure to 10 g/ml CSC in A549 while protein level peaked after 8h.
  • R mRNA induction correlated with interleukin-1 beta (IL- ⁇ ⁇ ) mRNA induction. It did not correlate with mRNA levels of tumor necrosis factor-alpha (TNF-a) and B 2 R was hardly affected by CSC treatment.
  • both B-i R protein and mRNA were maximally induced after 24h exposure to 5 pg/ml CSC in LS.
  • BiR represents a useful biomarker of inflammation related to cigarette smoke exposure.
  • HEF Human lung fibroblasts
  • A549) human alveolar epithelial cell lines
  • HLF and A549 purchased from ATCC, were cultured in F12 medium (Gibco) supplemented with 10% fetal calf serum and antibiotics (penicillin plus streptomycin). Cultured cells were maintained at 37°C with 5% C0 2 in a humified incubator. At their exponential growth phase, cells were exposed to IL- ⁇ and different concentrations of CSC for the designated durations; note that DMSO in all CSC treated groups was adjusted to the same level as highest CSC group including the DMSO control group. At the end of the treatment, cells were washed with PBS and lysed by Qiazol (Qiagen). Cell lysates were then stored in -80 °C before RNA extraction.
  • F12 medium Gibco
  • antibiotics penicillin plus streptomycin
  • TPM total particulate matter
  • cigarettes were conditioned and smoked under the environmental conditions specified in ISO 3042 ( 999) "Tobacco and tobacco products - Atmosphere for conditioning and testing”.
  • TPM was determined according to International Standard ISO 4387 Cigarettes - Determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine (Reference number ISO4387:2000; see also: Health Canada, "Determination of "Tar”, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke", 1999).
  • Detailed information on the collection of TPM is described in Appendix 1 of the Health Canada Official Method T-501 , Bacterial Reverse Mutation Assay for Mainstream Tobacco Smoke.
  • the lung was removed under aseptic conditions and incubated at 4°C for 20 minutes then cut into 0.5 mm thick longitudinal sections with a Stadie-Riggs tissue slicer (Thomas Scientific, Swedesboro, NJ, USA) at room temperature under an aseptic hood.
  • Lung sections (provided by UdeM) were kept in 50 ml bottle containing RPMI culture medium and transported on ice (4°C) to laboratories where they were incubated and treated. Lung sections were further cultured with RPMI 1640 (Gibco) medium overnight at 37°C before treatment; the details of culture condition were previously described (Lin et al., 1998).
  • RNAIater Qiagen
  • QRT-PCR quantitative real-time polymerase chain reaction
  • PCR conditions were as follows: 95°C for 15 min, followed by 46 cycles at 94°C for 15 s, 60°C for 30 s and 72°C for 30 s.
  • the cycle threshold (Ct) value represents the cycle number at which a fluorescent signal rises statistically above background (Wada et al., 2000).
  • the relative quantification of gene expression was analyzed by the 2- ct method (Livak & Schmittgen, 2001). RNA quality was assessed by melting.
  • Rat lung slices and human cells exposed to CSC were harvested and frozen at -80°C. Tissues and cells were resuspended in sterile phosphate buffered saline (PBS) containing a cocktail of proteases inhibitors (Sigma- Aldrich Canada), homogenised using polytron and centrifuged at 500 g. Supernatants were recovered and protein concentration was determined by the bicinchoninic acid method (BCA method) (Pierce).
  • PBS sterile phosphate buffered saline
  • BCA method bicinchoninic acid method
  • the antibodies were highly specific as demonstrated by the appearance of a band at 37 kDa or 42 kDa (putative molecular weight of rat and human B-
  • specificity was also confirmed by the absence of the 37 kDa band in mice invalidated for B1 R gene (KOB1 R mice) tissues.
  • the C-terminal epitope sequence for human anti B1 R is ISSSHRKEIFQLFWRN (16AA) and the C-terminal epitope sequence for rat anti B R is VFAGRLLKTRVLGTL (15AA). Smaller fragments, such as at least 5 amino acids, are also useful epitopes.
  • the secondary antibody for B-iR identification was an horseradish peroxydase (HRP)-linked goat anti-rabbit (Santa Cruz Biotech, CA) used at a 1 :25000 dilution. All incubations with antibodies were performed in phosphate buffered saline (PBS)-Tween 20 with milk. Membranes were rinsed adequately between every step with PBS-Tween 20, and Bi receptor protein revealed using enhanced chemiluminescence detection kit (Super-Signal, Pierce, Canada). Dynein was used as standard protein and revealed with mouse anti-dynein monoclonal antibody (Santa Cruz Biotech, CA) at a 1 :20000 dilution. HRP- linked goat anti-mouse (Santa Cruz Biotech, CA) was used as secondary antibody at a 1 :25000 dilution.
  • HRP- linked goat anti-mouse Santa Cruz Biotech, CA
  • Lung slices stored at -80°C were mounted in a gelatin block and serially cut into 20 ⁇ thick coronal sections (20 m x 1 mm length) on a cryostat at - 3°C. Sections were put on slides for one month at -80°C to allow adequate adhesion to the glass. Agarose retained in the lung sections was eliminated by pre-incubation in 25 mM PIPES buffer (pH 7.4) at room temperature for 10 min. The slides were transferred to 25 mM PIPES buffer containing peptidase inhibitors and the B-
  • BODIPY-des- Arg 9 -Bradykinine BODIPY-des- Arg 9 -Bradykinine
  • IL-1 p 50 pg/ml; 24h
  • IL- ⁇ also increased TNF-a expression in HLF.
  • CSC at 5 pg/ml had no effect on B ⁇ expression, but at 12.5 pg/ml it induced maximal B-
  • TNF-a mRNA induction was not correlated with BiR induction and IL-1 ⁇ expression.
  • the effect of CSC on B ⁇ , IL-1 ⁇ or TNF-a gene induction in HLF was not observed.
  • IL-1 ⁇ produced the same effect as DMSO on BiR protein expression in A549 (1.007 fold) and HLF (1.048 fold).
  • CSC at 12.5 and 30 pg/ml increased slightly BiR protein expression in A549 but had no effect in HLF.
  • a basal level of B-iR, IL- ⁇ and TNF-a mRNA was detected in DMSO-treated groups and untreated groups.
  • IL- ⁇ -treatment produced a maximal induction of B-iR, IL- ⁇ ⁇ and TNF-a mRNA following 4 h incubation.
  • Time-course experiments revealed that cells exposure to 10 ⁇ g/ml CSC for four 4h produced the maximal upregulation of BiR and [L-1 ⁇ mRNA, while TNF-a mRNA level was not affected by the same treatment regimen. Results are illustrated in Figure 2A.
  • a basal level of BiR, IL-1 ⁇ and TNF-a mRNA was detected in untreated groups.
  • the concentration of 10 ⁇ ig/m ⁇ of smoke condensate produced a maximal and significant upregulation of B-
  • IL-1 R antagonist, SN-50 and NAC showed strong and Pentox showed partial inhibitory effect of CSC-induced BiR expression, whereas capsazepine, niflumic acid and indomethacin failed in such total or partial inhibitory effect.
  • all inhibitors appeared to down regulate the basal level expression of IL-1 ⁇ and B-
  • BdABK BODIPY-des-Arg 9 -Bradykinin
  • Colocalisation of BiR on infiltrating neutrophils was noted in cultured rat lung slices treated with smoke condensate. Staining was identified representing positive B-
  • Colocalisation of BiR on alveolar epithelial cells was identified in cultured rat lung slices treated with smoke condensate. Staining represented positive BiR distribution throughout the cultured lung tissue exposed to CSC. Other staining selectively labelled alveolar epithelial cells. Colocalisation of B-
  • BiR expression was assessed in the lungs and trachea of rats exposed by inhalation to different cigarette smoke condensates using RNA extraction from formalin-fixed, paraffin-embedded material.
  • the inventors used the following procedures to assess kinin B1 receptor as a biomarker of inflammation related to cigarette smoke exposure in the trachea:
  • Paraffin-embedded tissue blocks were cut with a microtome into 15 pm sections (10-15 mg of tissue) and placed in RNase-free Eppendorf tubes. Tissues were deparaffinized by incubation in two consecutive baths of xylene for 5 min each, then in two consecutive baths of 100% ethanol for 5 min each. After deparaffinization and centrifugation, the pellets were air-dried (Gloghini et al., 2004).
  • Real-time quantitative PCR was performed using a Rotor Gene 6000 series (Corbett Life Science).
  • the system automatically monitors the binding of a fluorescent dye SYBR R Green to double- stranded DNA by real-time detection of the fluorescence during each cycle of PCR amplification. After 2 min at 50°C for UNG incubation and 10 min at 95°C for polymerase activation, 40 cycles of 15 s at 95°C (denaturation) and 1 min at 60°C (annealing and extension) were run. Only those reactions which gave a well defined amplification product both by melting curve analysis and agarose gel electrophoresis were included in the quantitative analysis. All the PCR primers were designed by using Prime Express 2.0 software (Applied Biosystem, USA) and synthesized by MEDICORP Inc. (Montreal, Canada). The sequences are:
  • the 2 " C T method (Livak and Schmittgen, 2001) was employed to calculate relative changes in gene expression for BiR between treatments.
  • the threshold cycle (C T ) for housekeeping gene ⁇ -actin was used as reference to normalize a relative amount of mRNA for B-
  • the ⁇ C T was calculated for each treatment (C T B-iR - C T ⁇ -actin) and the value of untreated control (CT BiR - CT ⁇ -actin) was subtracted to yield Cj.
  • Realtime PCR was made on three replicates for each sample and a blank control (no template) was included in all the experiments for negative controls.
  • RNAs were extracted from tissue according to the manufacturer's protocol.
  • First-strand cDNA synthesized from 400 ng total RNA with random hexamer primers was used as template for each reaction with the QuantiTect Rev Transcription Kit (QIAGEN).
  • SYBR Green-based real-time quantitative PCR was performed as described (Aoki et al., 2002).
  • Mx3000p (Stratagene) was used for the signal detection and the PCR was performed in SYBR Green Master mix (QIAGEN) with 300 nM of each primer.
  • PCR conditions were: 95°C for 15 min, followed by 46 cycles at 94°C for 15 s, 60°C for 30 s and 72°C for 30 s.
  • the cycle threshold (Ct) value represents the cycle number at which a fluorescent signal rises statistically above background (Wada et al., 2000).
  • the relative quantification of gene expression was analyzed by the 2-ACt method (Livak & Schmittgen, 2001).
  • the housekeeping gene 18S was used as reference to normalize a relative amount of mRNA for BiR in the same sample. Standard curves with 8S and the conditions of amplification were generated.
  • the following primer pairs were designed by Vector NTI and used:
  • the inventors show the usefulness of kinin B1 receptor as a buccal epithelial cell biomarker of inflammation for concentration-dependent induction of E ⁇ R RNA and protein after CSC treatment.
  • the inventors use buccal cells collected from human volunteers and rats.
  • BiR mRNA and protein expression is also shown in mammalian mouth tissue following treatment with CSC. Time dependency of induction is also readily demonstrated.
  • BiR represents a useful buccal epithelial cell biomarker of inflammation related to cigarette smoke exposure.
  • Cells are collected by either scraping the buccal mucosa (El- Setouhy et al. 2008; Sridhar et al. 2008) or via a mouthwash protocol (Heath et al. 2001). Cells are maintained at 37°C with C0 2 in a humidified incubator. If needed, cells are exposed to IL-1 ⁇ and/or different concentrations of CSC as previously described for A549 cells. At the end of the treatment, cells are washed and stored before RNA extraction. For Western blot analysis, cells are washed and collected by centrifugation. Cell lysates are subjected to PCR analysis of BiR or for the Western blot analysis, B-
  • RNA is extracted from cells and subjected to PCR as noted above for lung PCR. Primer sequences are designed by Vector NTI software. The results show that CSC increases BiR RNA expression.
  • the invention validates kinin Bi receptor as a biomarker of inflammation induced by smoke exposure.
  • the inventors showed Bi upregulation in experiments in human epithelial cell lines (e.g. A549) and cultured rat lung slices (LS).
  • Human cell lines were used to demonstrate that BiR was induced in cultured human cells exposed to CSC.
  • Rat lung slices were used as suitable ex vivo model to localise the BiR because it has the advantages to maintain cell-matrix interaction and the immune cell function which can contribute in the induction of BT R by CSC.
  • the human cell lines were exposed to various concentrations of CSC, vehicle (DMSO) and interleukin- ⁇ (positive control). Harvesting these cells at different time points allowed the inventors to establish time- and concentration-response curves on the expression of BiR, B 2 R and proinflammatory cytokines (IL- ⁇ and TNF-a).
  • cell lines exposed or not to CSC and to various inhibitors namely capsazepine (TRPV1 antagonist), N- acetyl-L-cysteine (ROS scavenger), pentoxifylin (inhibitor of TNF-a release), indomethacin (COX inhibitor), niflumic acid (COX-2 inhibitor), SN-50 (NF-(B inhibitor), IL-1 Ra (interleukin-1 receptor antagonist) allowed the inventors to determine the mechanism implicated in B ⁇ R upregulation.
  • TRPV1 antagonist capsazepine
  • ROS scavenger N- acetyl-L-cysteine
  • pentoxifylin inhibitor of TNF-a release
  • indomethacin COX inhibitor
  • COX-2 inhibitor niflumic acid
  • SN-50 NF-(B inhibitor)
  • IL-1 Ra interleukin-1 receptor antagonist
  • A549 was a better model than HLF to induce B-
  • R mRNA and protein
  • CSC had no effect on BiR, IL-1 ⁇ and TNF-a mRNA in these cells. Based on these results, the inventors decided to pursue their next experiments in A549.
  • the inventors then defined the best concentrations of CSC to achieve maximal BiR induction by CSC in A549.
  • IL-1 ⁇ was able to upregulate B-iR mRNA in A549 cell line.
  • R but not B 2 R was induced when exposed to CSC and this upregulation was maximal at 0 pg/ml of CSC.
  • B ⁇ mRNA induction correlated strongly with 1L-1 ⁇ induction but not with TNF-a which was not consistently induced.
  • LS tissue may respond to lower concentration of CSC because it involves many cell types that could participate in a synergistic manner to induction.
  • TNF-a mRNA level correlated with B ⁇ induction only in LS. This observation can be explained by the fact that some specific cell types of LS could be more reactive to TNF-a or could release more of this cytokine by opposition to A549 cell line, which is more dependent on IL-1 ⁇ for B-
  • BdABK Bodipy-des-Arg 9 -Bk
  • epithelial cells have a critical role in the generation of lung diseases.
  • Cigarette smoke is a risk factor in the development of various lung diseases including pulmonary emphysema, pulmonary fibrosis, and lung cancer.
  • the mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following exposure to cigarette smoke, alterations in alveolar epithelial cells increased epithelial permeability, decreased surfactant production, caused an inappropriate production of inflammatory cytokines and growth factors, and increased risk of lung cancer.
  • control refers to an experimental comparison group tested in the same manner as the experimental sample except for a variable(s) of interest that is being tested in the experimental sample.
  • Controls in the present application may be negative controls, such as cells, tissues or organs that have not been exposed to a test substance. For example, if one is testing CSC, the control group may remain unexposed to CSC. If mammals, organs or tissues are tested with smoke as a test substance, the control group is optionally non-smokers. Controls may also be positive controls, such as cells, tissues or organs that have not been exposed to certain compounds, such as IL-1 ⁇ that induce inflammation, but not to a test substance.
  • a control is optionally a pre-determined standardized result, for example, biomarker levels, numerical values and/or ranges (e.g. control range) corresponding to the biomarker level for a typical cell, tissue or organ when it is either in an inflamed state or in a non-inflamed state.
  • the result may be a pre-determined, non- inflamed standard result or a pre-determined, inflamed standard result. For example, it is not required to run an experimental comparison group alongside every experimental sample when a pre-determined standardized result is available.
  • a control population may also be used, for example if comparing smoker (smoke-contacted) samples to a control population of non-smokers.
  • R refers to measuring or detecting the expression of B-
  • the determining step may be qualitative or quantitative.
  • hybridize refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid.
  • One aspect of the invention provides an isolated nucleotide sequence, which hybridizes to a RNA product of a gene encoding a protein of interest or a nucleic acid sequence which is complementary to an RNA product of a gene encoding a protein of interest.
  • the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. For example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C for 15 minutes or more may be employed.
  • SSC sodium chloride/sodium citrate
  • Inflammation in this application refers to the protective response of a cell, tissue or organ that is triggered by a foreign compound and mediated through a mechanism involving compounds such as cytokines, for example the pro-inflammatory cytokine IL-1 ⁇ .
  • the response involves release of inflammation-mediating chemicals from the cell, tissue or organ to defend against the foreign compound.
  • the chemicals have a number of downstream effects, such as recruiting immune cells, causing increased blood supply to an affected tissue or organ and swelling.
  • primer refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis of when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • biomarker specific primers refers a set of primers which can produce a double stranded nucleic acid product complementary to a portion of one or more RNA products of the biomarkers described herein or sequences complementary thereof.
  • probe refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence.
  • the probe hybridizes to a RNA product of the biomarker of the disclosure or a nucleic acid sequence complementary to the RNA product of the biomarker of the disclosure.
  • the length of probe depends on the hybridize conditions and the sequences of the probe and nucleic acid target sequence. In an embodiment, the probe is at least: 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length.
  • sample refers to any airway cell, tissue or organ specimen which is capable of expressing BiR and assayed for BiR expression.
  • sample is a lung, trachea or mouth cell that expresses BiR.
  • subject refers to any member of the animal kingdom, optionally a mammal, such as a human being.
  • A549 cultured human epithelial cell line
  • bradykinin B2 receptor bradykinin B2 receptor
  • BdABK BODIPY-des-Arg 9 -Bradykinin
  • CGRP calcitonin gene-related peptide
  • COPD chronic obstructive pulmonary disease
  • COX-1 cyclooxygenase-1
  • EpCAM epithelial cell adhesion molecule
  • GAPDH glyceraldehyde 3-phosphate dehydrogenase
  • HLF human lung fibroblasts
  • ⁇ _-1 ⁇ interleukin-1 beta
  • IL-1 Ra IL-1 R antagonist
  • KOB1 R mice invalidated for B1 R gene
  • LPS bacterial lipopolysaccharid
  • MAPK mitogen-activated protein kinase
  • mRNA messenger ribonucleic acid
  • NAC N-acetyl-L-cysteine
  • NF-KB nuclear transcription factor-kappa B
  • PBS phosphate buffered saline
  • TRPV1 transient receptor potential vanilloid 1
  • ARAUJO RC KETTRITZ R, FICHTNER I, PAIVA AC, PESQUERO JB, BADER M. (2001).
  • GUENTER CA COALSON JJ, JACQUES J. (1981). Emphysema associated with intravascular leukocyte sequestration. Comparison with papain-induced emphysema. Am Rev Respir Dis. 123:79-84.
  • Bradykinin B1 receptor up-regulation by interleukin- 1 beta and B1 agonist occurs through independent and synergistic intracellular signaling mechanisms in human lung fibroblasts. J Pharmacol Exp Ther. 298:77-85.
  • PROUD D (1998). The kinin system in rhinitis and asthma. Clin Rev Allergy lmmunol.16:351-64.
  • Aerosolized human neutrophil elastase induces airway constriction and hyperresponsiveness with protection by intravenous pretreatment with half- length secretory leukoprotease inhibitor. Am J Respir Crit Care Med. 153:1405- 11.
  • CHOMCZYNSKI P.A. (1993). A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15: 532-4, 536-7.
  • ONGALI B., CAMPOS, M.M., PETCU, M., RODI, D., CLOUTIER, F., CHABOT, J.-G., THIBAULT, G., COUTURE, R. (2004). Expression of kinin receptors in the spinal cord of streptozotocin-diabetic rat. Neuroreport 15: 2463-2466. PLANELLS-CASES, R., GARCIA-SAN, N., MORENILLA-PALOA, C, FERRER- MONTIEL, A. (2005). Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia. Plugers Arch-Eur. J. Pharmacol. 451 : 151-159.
  • Heath EM Morken NW, Campbell KA, Tkach D, Boyd EA, (2001). "Use of buccal cells collected in mouthwash as a source of DNA for clinical testing.” Arch Pathol Lab Med 125(1): 127-33.

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Abstract

L'invention concerne un procédé de détermination du fait qu'une substance test provoque une inflammation des cellules des voies aériennes, le procédé comprenant : la mise en contact de la substance test avec un échantillon comprenant une cellule des voies aériennes exprimant le récepteur de la bradykinine B1 (B1R); et la détermination de si ou non le niveau d'expression de B1R dans la cellule est augmenté significativement en présence de la substance, indiquant par là que la substance provoque une inflammation. Le niveau de signification de l'augmentation de l'expression de B1R est typiquement basé sur une comparaison avec le niveau de B1R dans la cellule avant l'exposition à la substance.
PCT/EP2010/068348 2009-11-27 2010-11-26 Récepteur de quinine b1 comme biomarqueur d'une inflammation des voies aériennes liée à une exposition à la fumée Ceased WO2011064351A1 (fr)

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CN102384961A (zh) * 2011-10-16 2012-03-21 云南烟草科学研究院 添加剂对卷烟特殊烟气指标影响程度的判别方法
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CN102384961B (zh) * 2011-10-16 2014-06-11 云南烟草科学研究院 添加剂对卷烟特殊烟气指标影响程度的判别方法
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