Limits...
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.

Show MeSH

Related in: MedlinePlus

Specific Reduction of pSTAT3, but not pSTAT5 in Dexamethasone Treated CAIA Mice Overtime Corresponds with Local Dexamethasone Pharmacokinetics. Using our CIA mouse model of RA we treated 35 day mice with either vehicle alone or dexamethasone and looked overtime at the loss of pSTAT3 and pSTAT5 in the paws using our extraction method. (A) Paws from treated mice were processed and analyzed using Luminex® technology, rapid decrease in pSTAT3 observed within 4 hours post dexamethasone treatment and remains decreased as shown for the 6 hour time point compared to vehicle alone, *p < 0.05. (B) No significant change observed for pSTAT5, trend decrease in overall pSTAT5 levels, ns = not significant as compared to vehicle alone. (C) PK of dexamethasone in the plasma, spleen and paw extracts from treated mice, see materials and methods for complete methods for PK analysis. All graphs show Mean ± SEM, N = 4 mice per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2992046&req=5

Figure 5: Specific Reduction of pSTAT3, but not pSTAT5 in Dexamethasone Treated CAIA Mice Overtime Corresponds with Local Dexamethasone Pharmacokinetics. Using our CIA mouse model of RA we treated 35 day mice with either vehicle alone or dexamethasone and looked overtime at the loss of pSTAT3 and pSTAT5 in the paws using our extraction method. (A) Paws from treated mice were processed and analyzed using Luminex® technology, rapid decrease in pSTAT3 observed within 4 hours post dexamethasone treatment and remains decreased as shown for the 6 hour time point compared to vehicle alone, *p < 0.05. (B) No significant change observed for pSTAT5, trend decrease in overall pSTAT5 levels, ns = not significant as compared to vehicle alone. (C) PK of dexamethasone in the plasma, spleen and paw extracts from treated mice, see materials and methods for complete methods for PK analysis. All graphs show Mean ± SEM, N = 4 mice per group.

Mentions: Using the CAIA model significant decrease in the level of pSTAT3 as early as 4 hours after dexamethasone treatment (Figure 5A) could be observed, however no significant change in pSTAT5a/b levels were ever observed, albeit a trend decrease does support the case of dexamethasone acting as a general immunosuppressant (Figure 5B). As shown by pharmacokinetic analysis, dexamethasone can be detected in the paw as early as 2 hours post-injection and can affect downstream effector cascades (Figure 5C). This suggests that similar therapies can be used with this reference standard of care (SoC) agent to monitor cytokine pathway modulation in conjunction with disease as exemplified in Figures 2.0 and 3.0. Similar pSTAT results were also obtained for the CIA model (Additional file 5, Figure S5) for both pSTAT3 and pSTAT5, no change in pSTAT1 was observed in any model tested (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)

Specific Reduction of pSTAT3, but not pSTAT5 in Dexamethasone Treated CAIA Mice Overtime Corresponds with Local Dexamethasone Pharmacokinetics. Using our CIA mouse model of RA we treated 35 day mice with either vehicle alone or dexamethasone and looked overtime at the loss of pSTAT3 and pSTAT5 in the paws using our extraction method. (A) Paws from treated mice were processed and analyzed using Luminex® technology, rapid decrease in pSTAT3 observed within 4 hours post dexamethasone treatment and remains decreased as shown for the 6 hour time point compared to vehicle alone, *p < 0.05. (B) No significant change observed for pSTAT5, trend decrease in overall pSTAT5 levels, ns = not significant as compared to vehicle alone. (C) PK of dexamethasone in the plasma, spleen and paw extracts from treated mice, see materials and methods for complete methods for PK analysis. All graphs show Mean ± SEM, N = 4 mice per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992046&req=5

Figure 5: Specific Reduction of pSTAT3, but not pSTAT5 in Dexamethasone Treated CAIA Mice Overtime Corresponds with Local Dexamethasone Pharmacokinetics. Using our CIA mouse model of RA we treated 35 day mice with either vehicle alone or dexamethasone and looked overtime at the loss of pSTAT3 and pSTAT5 in the paws using our extraction method. (A) Paws from treated mice were processed and analyzed using Luminex® technology, rapid decrease in pSTAT3 observed within 4 hours post dexamethasone treatment and remains decreased as shown for the 6 hour time point compared to vehicle alone, *p < 0.05. (B) No significant change observed for pSTAT5, trend decrease in overall pSTAT5 levels, ns = not significant as compared to vehicle alone. (C) PK of dexamethasone in the plasma, spleen and paw extracts from treated mice, see materials and methods for complete methods for PK analysis. All graphs show Mean ± SEM, N = 4 mice per group.
Mentions: Using the CAIA model significant decrease in the level of pSTAT3 as early as 4 hours after dexamethasone treatment (Figure 5A) could be observed, however no significant change in pSTAT5a/b levels were ever observed, albeit a trend decrease does support the case of dexamethasone acting as a general immunosuppressant (Figure 5B). As shown by pharmacokinetic analysis, dexamethasone can be detected in the paw as early as 2 hours post-injection and can affect downstream effector cascades (Figure 5C). This suggests that similar therapies can be used with this reference standard of care (SoC) agent to monitor cytokine pathway modulation in conjunction with disease as exemplified in Figures 2.0 and 3.0. Similar pSTAT results were also obtained for the CIA model (Additional file 5, Figure S5) for both pSTAT3 and pSTAT5, no change in pSTAT1 was observed in any model tested (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