Evaluation of salivary cytokines for diagnosis of both trauma-induced and genetic heterotopic ossification

Hsiao Hsin Sung Hsieh, Michael T. Chung, Ronald M. Allen, Kavitha Ranganathan, Joe Habbouche, David Cholok, Jonathan Butts, Arminder Kaura, Ramkumar Tiruvannamalai-Annamalai, Chris Breuler, Caitlin Priest, Shawn J. Loder, John Li, Shuli Li, Jan Stegemann, Steven L. Kunkel, Benjamin Levi

Research output: Contribution to journalArticle

Abstract

Purpose: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. Methods: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. Results: Levels of TNF-a and IL-1β peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1β, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). Conclusion: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.

Original languageEnglish (US)
Article number74
JournalFrontiers in Endocrinology
Volume8
Issue numberAPR
DOIs
StatePublished - Apr 24 2017
Externally publishedYes

Fingerprint

Heterotopic Ossification
Cytokines
Tenotomy
Wounds and Injuries
Chemokine CCL2
Biomarkers
Burns
Saliva
Vascular Endothelial Growth Factor A
Serum
Interleukin-1
Myositis Ossificans
Salivary Proteins and Peptides
Fluorescent Antibody Technique
Blood Proteins
Real-Time Polymerase Chain Reaction
Early Diagnosis
Extremities
Staining and Labeling
Inflammation

Keywords

  • Biomarkers
  • Fibrodysplasia ossificans progresiva
  • Heterotopic ossification
  • Inflammatory cytokines
  • Minimally invasive
  • Saliva

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism

Cite this

Evaluation of salivary cytokines for diagnosis of both trauma-induced and genetic heterotopic ossification. / Hsieh, Hsiao Hsin Sung; Chung, Michael T.; Allen, Ronald M.; Ranganathan, Kavitha; Habbouche, Joe; Cholok, David; Butts, Jonathan; Kaura, Arminder; Tiruvannamalai-Annamalai, Ramkumar; Breuler, Chris; Priest, Caitlin; Loder, Shawn J.; Li, John; Li, Shuli; Stegemann, Jan; Kunkel, Steven L.; Levi, Benjamin.

In: Frontiers in Endocrinology, Vol. 8, No. APR, 74, 24.04.2017.

Research output: Contribution to journalArticle

Hsieh, HHS, Chung, MT, Allen, RM, Ranganathan, K, Habbouche, J, Cholok, D, Butts, J, Kaura, A, Tiruvannamalai-Annamalai, R, Breuler, C, Priest, C, Loder, SJ, Li, J, Li, S, Stegemann, J, Kunkel, SL & Levi, B 2017, 'Evaluation of salivary cytokines for diagnosis of both trauma-induced and genetic heterotopic ossification', Frontiers in Endocrinology, vol. 8, no. APR, 74. https://doi.org/10.3389/fendo.2017.00074
Hsieh, Hsiao Hsin Sung ; Chung, Michael T. ; Allen, Ronald M. ; Ranganathan, Kavitha ; Habbouche, Joe ; Cholok, David ; Butts, Jonathan ; Kaura, Arminder ; Tiruvannamalai-Annamalai, Ramkumar ; Breuler, Chris ; Priest, Caitlin ; Loder, Shawn J. ; Li, John ; Li, Shuli ; Stegemann, Jan ; Kunkel, Steven L. ; Levi, Benjamin. / Evaluation of salivary cytokines for diagnosis of both trauma-induced and genetic heterotopic ossification. In: Frontiers in Endocrinology. 2017 ; Vol. 8, No. APR.
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abstract = "Purpose: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. Methods: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. Results: Levels of TNF-a and IL-1β peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1β, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). Conclusion: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.",
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T1 - Evaluation of salivary cytokines for diagnosis of both trauma-induced and genetic heterotopic ossification

AU - Hsieh, Hsiao Hsin Sung

AU - Chung, Michael T.

AU - Allen, Ronald M.

AU - Ranganathan, Kavitha

AU - Habbouche, Joe

AU - Cholok, David

AU - Butts, Jonathan

AU - Kaura, Arminder

AU - Tiruvannamalai-Annamalai, Ramkumar

AU - Breuler, Chris

AU - Priest, Caitlin

AU - Loder, Shawn J.

AU - Li, John

AU - Li, Shuli

AU - Stegemann, Jan

AU - Kunkel, Steven L.

AU - Levi, Benjamin

PY - 2017/4/24

Y1 - 2017/4/24

N2 - Purpose: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. Methods: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. Results: Levels of TNF-a and IL-1β peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1β, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). Conclusion: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.

AB - Purpose: Heterotopic ossification (HO) occurs in the setting of persistent systemic inflammation. The identification of reliable biomarkers can serve as an early diagnostic tool for HO, especially given the current lack of effective treatment strategies. Although serum biomarkers have great utility, they can be inappropriate or ineffective in traumatic acute injuries and in patients with fibrodysplasia ossificans progressiva (FOP). Therefore, the goal of this study is to profile the cytokines associated with HO using a different non-invasive source of biomarkers. Methods: Serum and saliva were collected from a model of trauma-induced HO (tHO) with hind limb Achilles' tenotomy and dorsal burn injury at indicated time points (pre-injury, 48 h, 1 week, and 3 weeks post-injury) and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt). Samples were analyzed for 27 cytokines using the Bio-Plex assay. Histologic evaluation was performed in Nfatc1-Cre/caAcvr1fl/wt mice and at 48 h and 1 week post-injury in burn tenotomy mice. The mRNA expression levels of these cytokines at the tenotomy site were also quantified with quantitative real-time PCR. Pearson correlation coefficient was assessed between saliva and serum. Results: Levels of TNF-a and IL-1β peaked at 48 h and 1 week post-injury in the burn/tenotomy cohort, and these values were significantly higher when compared with both uninjured (p < 0.01, p < 0.03) and burn-only mice (p < 0.01, p < 0.01). Immunofluorescence staining confirmed enhanced expression of IL-1β, TNF-α, and MCP-1 at the tenotomy site 48 h after injury. Monocyte chemoattractant protein-1 (MCP-1) and VEGF was detected in saliva showing elevated levels at 1 week post-injury in our tHO model when compared with both uninjured (p < 0.001, p < 0.01) and burn-only mice (p < 0.005, p < 0.01). The Pearson correlation between serum MCP-1 and salivary MCP-1 was statistically significant (r = 0.9686, p < 0.001) Similarly, the Pearson correlation between serum VEGF and salivary VEGF was statistically significant (r = 0.9709, p < 0.05). Conclusion: In this preliminary study, we characterized the diagnostic potential of specific salivary cytokines that may serve as biomarkers for an early-stage diagnosis of HO. This study identified two candidate biomarkers for further study and suggests a novel method for diagnosis in the context of current difficult diagnosis and risks of current diagnostic methods in certain patients.

KW - Biomarkers

KW - Fibrodysplasia ossificans progresiva

KW - Heterotopic ossification

KW - Inflammatory cytokines

KW - Minimally invasive

KW - Saliva

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