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Novel method of monitoring trace cytokines and activated STAT molecules in the paws of arthritic mice using multiplex bead technology.

Lu LD, Stump KL, Seavey MM - BMC Immunol. (2010)

Bottom Line: How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained.Local cytokine responses could be matched with serum cytokine levels and joint pathology results.STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Worldwide Discovery Research, Cephalon, Inc, West Chester, Pennsylvania 19380, USA.

ABSTRACT

Background: The use of mouse models to study human disease provides useful data that can provide support for research projects or an existing drug discovery program. How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained. Designing new treatments for rheumatic diseases, such as rheumatoid arthritis (RA), requires complex immunocompetent models that depend on intricate cytokine networks. Using local cytokines, signal transduction and transcription factor molecules as potential biomarkers to monitor disease and treatment efficacy is the best method to follow the progression of tissue damage and repair when testing an unknown compound or biologic. Described here in this report, a novel method for the non-enzymatic extraction and measurement of cytokines and signal transducers and activators of transcription (STAT) molecules using Luminex® bead array technology in two different mouse models for human RA--collagen antibody-dependent arthritis (CAIA) and collagen-induced arthritis (CIA).

Results: Dynamic expression of several pro-inflammatory cytokines responsible for promoting disease augmentation overtime were monitored, such as IL-1β, TNFα, IL-6 and IL-12, locally in the paws of affected animals directly ex vivo. Local cytokine responses could be matched with serum cytokine levels and joint pathology results. In addition, STAT1, 3, and 5a/b activation status could be monitored with confidence using specifically formulated extraction buffer that protected the phosphorylation site. STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone. Here reported a novel method of extracting joint fluid from the paws of inflamed mice coupled with powerful multiplex bead technology allowing us to measure cytokine responses, pharmacodynamic markers such as STATs and pharmacokinetic analysis of dosed agent all from the same sample directly ex vivo.

Conclusions: This method is powerful in that it is applicable to multiple autoimmunity model types, streamlines ex vivo readouts in a high-throughput manner, and allows multiplexing providing the investigator with an array of options and possible analytes when developing preclinical animal models to support drug discovery efforts in the search for new treatments for rheumatic diseases.

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Using Luminex® Multiplex Bead Arrays to Measure Intracellular PD Markers is More Sensitive than Conventional Western Blot Analysis. Using the 60 day CIA mouse paws we extracted the paw supernatants as described in the materials and methods and tested for phospho-STAT3 using either conventional western blot analysis with densitometry or multiplex bead analysis. (A) Extracts from two different mice per group were run on a SDS-PAGE gel and transferred onto a nitrocellulose membrane and exposed to film for the final image. Western blot was probed with anti-pSTAT3 α (79 kDa) and β (86 kDa) subunits, stripped then re-probed for total STAT3, α (76 kDα) and β (86 kDa) subunits as shown. Below graph shows densitometry analysis from above blot **ns p = 0.2037 versus vehicle, *ns p = 0.1226 versus CIA + vehicle group, N = 3 per group shown. (B) Luminex® phospho-STAT kit analyzed paw extractions from CIA mice treated with vehicle alone or dexamethasone. Samples were those same as used for the W.blot above, *p = 0.034 compared CIA + vehicle group, **p = 0.017 compared to vehicle alone. For both graphs, statistical test used for p-values: two-tailed paired Student's t-test, N = 3-4 mice per group shown. All graphs show Mean ± SEM, ns = not significant.
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Figure 4: Using Luminex® Multiplex Bead Arrays to Measure Intracellular PD Markers is More Sensitive than Conventional Western Blot Analysis. Using the 60 day CIA mouse paws we extracted the paw supernatants as described in the materials and methods and tested for phospho-STAT3 using either conventional western blot analysis with densitometry or multiplex bead analysis. (A) Extracts from two different mice per group were run on a SDS-PAGE gel and transferred onto a nitrocellulose membrane and exposed to film for the final image. Western blot was probed with anti-pSTAT3 α (79 kDa) and β (86 kDa) subunits, stripped then re-probed for total STAT3, α (76 kDα) and β (86 kDa) subunits as shown. Below graph shows densitometry analysis from above blot **ns p = 0.2037 versus vehicle, *ns p = 0.1226 versus CIA + vehicle group, N = 3 per group shown. (B) Luminex® phospho-STAT kit analyzed paw extractions from CIA mice treated with vehicle alone or dexamethasone. Samples were those same as used for the W.blot above, *p = 0.034 compared CIA + vehicle group, **p = 0.017 compared to vehicle alone. For both graphs, statistical test used for p-values: two-tailed paired Student's t-test, N = 3-4 mice per group shown. All graphs show Mean ± SEM, ns = not significant.

Mentions: To compare our multiplex bead method to the conventional western blot analysis we ran inflamed paw extracts from CAIA mice on a western blot side-by-side with the multiplex bead assay measuring phosphorylated STAT1 (pSTAT1), STAT3 (pSTAT3) and STAT5a/b (pSTAT5a/b). Both pSTAT1 and pSTAT5a/b were similar to the vehicle only group (data not shown), however, pSTAT3 showed a dramatic change from vehicle control group and this was similar with results seen for the western blot (Figure 4A-B; only pSTAT3 data shown). Lanes represent two independent samples from each group, densitometry analysis results are shown below the blot. Comparing these results (Figure 4A) to the multiplex bead results (Figure 4B) the bead array provides better data resolution and a lower and more reproducible background. Although, results are comparable between the two methods, the Luminex® data provides greater statistical significance due to the larger signal to noise ratio. However, the bead array provides the ability to simultaneously measure several different STAT molecules in a single high-throughput manner. Similar results were observed for the spleen tissue but were not as robust as the paw where most of the disease activity is confined (data not shown).


Novel method of monitoring trace cytokines and activated STAT molecules in the paws of arthritic mice using multiplex bead technology.

Lu LD, Stump KL, Seavey MM - BMC Immunol. (2010)

Using Luminex® Multiplex Bead Arrays to Measure Intracellular PD Markers is More Sensitive than Conventional Western Blot Analysis. Using the 60 day CIA mouse paws we extracted the paw supernatants as described in the materials and methods and tested for phospho-STAT3 using either conventional western blot analysis with densitometry or multiplex bead analysis. (A) Extracts from two different mice per group were run on a SDS-PAGE gel and transferred onto a nitrocellulose membrane and exposed to film for the final image. Western blot was probed with anti-pSTAT3 α (79 kDa) and β (86 kDa) subunits, stripped then re-probed for total STAT3, α (76 kDα) and β (86 kDa) subunits as shown. Below graph shows densitometry analysis from above blot **ns p = 0.2037 versus vehicle, *ns p = 0.1226 versus CIA + vehicle group, N = 3 per group shown. (B) Luminex® phospho-STAT kit analyzed paw extractions from CIA mice treated with vehicle alone or dexamethasone. Samples were those same as used for the W.blot above, *p = 0.034 compared CIA + vehicle group, **p = 0.017 compared to vehicle alone. For both graphs, statistical test used for p-values: two-tailed paired Student's t-test, N = 3-4 mice per group shown. All graphs show Mean ± SEM, ns = not significant.
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Figure 4: Using Luminex® Multiplex Bead Arrays to Measure Intracellular PD Markers is More Sensitive than Conventional Western Blot Analysis. Using the 60 day CIA mouse paws we extracted the paw supernatants as described in the materials and methods and tested for phospho-STAT3 using either conventional western blot analysis with densitometry or multiplex bead analysis. (A) Extracts from two different mice per group were run on a SDS-PAGE gel and transferred onto a nitrocellulose membrane and exposed to film for the final image. Western blot was probed with anti-pSTAT3 α (79 kDa) and β (86 kDa) subunits, stripped then re-probed for total STAT3, α (76 kDα) and β (86 kDa) subunits as shown. Below graph shows densitometry analysis from above blot **ns p = 0.2037 versus vehicle, *ns p = 0.1226 versus CIA + vehicle group, N = 3 per group shown. (B) Luminex® phospho-STAT kit analyzed paw extractions from CIA mice treated with vehicle alone or dexamethasone. Samples were those same as used for the W.blot above, *p = 0.034 compared CIA + vehicle group, **p = 0.017 compared to vehicle alone. For both graphs, statistical test used for p-values: two-tailed paired Student's t-test, N = 3-4 mice per group shown. All graphs show Mean ± SEM, ns = not significant.
Mentions: To compare our multiplex bead method to the conventional western blot analysis we ran inflamed paw extracts from CAIA mice on a western blot side-by-side with the multiplex bead assay measuring phosphorylated STAT1 (pSTAT1), STAT3 (pSTAT3) and STAT5a/b (pSTAT5a/b). Both pSTAT1 and pSTAT5a/b were similar to the vehicle only group (data not shown), however, pSTAT3 showed a dramatic change from vehicle control group and this was similar with results seen for the western blot (Figure 4A-B; only pSTAT3 data shown). Lanes represent two independent samples from each group, densitometry analysis results are shown below the blot. Comparing these results (Figure 4A) to the multiplex bead results (Figure 4B) the bead array provides better data resolution and a lower and more reproducible background. Although, results are comparable between the two methods, the Luminex® data provides greater statistical significance due to the larger signal to noise ratio. However, the bead array provides the ability to simultaneously measure several different STAT molecules in a single high-throughput manner. Similar results were observed for the spleen tissue but were not as robust as the paw where most of the disease activity is confined (data not shown).

Bottom Line: How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained.Local cytokine responses could be matched with serum cytokine levels and joint pathology results.STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone.

View Article: PubMed Central - HTML - PubMed

Affiliation: Worldwide Discovery Research, Cephalon, Inc, West Chester, Pennsylvania 19380, USA.

ABSTRACT

Background: The use of mouse models to study human disease provides useful data that can provide support for research projects or an existing drug discovery program. How well a model recapitulates the human condition and the ease and reproducibility of data collected will determine how much confidence a scientist can place on results obtained. Designing new treatments for rheumatic diseases, such as rheumatoid arthritis (RA), requires complex immunocompetent models that depend on intricate cytokine networks. Using local cytokines, signal transduction and transcription factor molecules as potential biomarkers to monitor disease and treatment efficacy is the best method to follow the progression of tissue damage and repair when testing an unknown compound or biologic. Described here in this report, a novel method for the non-enzymatic extraction and measurement of cytokines and signal transducers and activators of transcription (STAT) molecules using Luminex® bead array technology in two different mouse models for human RA--collagen antibody-dependent arthritis (CAIA) and collagen-induced arthritis (CIA).

Results: Dynamic expression of several pro-inflammatory cytokines responsible for promoting disease augmentation overtime were monitored, such as IL-1β, TNFα, IL-6 and IL-12, locally in the paws of affected animals directly ex vivo. Local cytokine responses could be matched with serum cytokine levels and joint pathology results. In addition, STAT1, 3, and 5a/b activation status could be monitored with confidence using specifically formulated extraction buffer that protected the phosphorylation site. STAT3 activation followed paw swelling and cytokine levels in both models and correlates of disease could be ablated upon treatment with dexamethasone. Here reported a novel method of extracting joint fluid from the paws of inflamed mice coupled with powerful multiplex bead technology allowing us to measure cytokine responses, pharmacodynamic markers such as STATs and pharmacokinetic analysis of dosed agent all from the same sample directly ex vivo.

Conclusions: This method is powerful in that it is applicable to multiple autoimmunity model types, streamlines ex vivo readouts in a high-throughput manner, and allows multiplexing providing the investigator with an array of options and possible analytes when developing preclinical animal models to support drug discovery efforts in the search for new treatments for rheumatic diseases.

Show MeSH
Related in: MedlinePlus