Multiplex serum protein analysis reveals potential mechanisms and markers of response to hyperimmune caprine serum in systemic sclerosis
- Niamh Quillinan†1,
- Kristina E. N. Clark†1,
- Bryan Youl2,
- Jeffrey Vernes3,
- Deirdre McIntosh3,
- Syed Haq3 and
- Christopher P. Denton1Email author
© The Author(s). 2017
Received: 6 September 2016
Accepted: 1 February 2017
Published: 7 March 2017
Hyperimmune caprine serum (HICS) is a novel biological therapy with potential benefit for skin in established diffuse cutaneous systemic sclerosis. Here we report multiplex protein analysis of blood samples from a placebo-controlled phase II clinical trial and explore mechanisms of action and markers of response.
Patients were treated with HICS (n = 10) or placebo (n = 10) over 26 weeks, with follow-up open-label treatment to 52 weeks in 14 patients. Serum or plasma samples at baseline, 26 and 52 weeks were analysed using multiplex or individual immunoassays for 41 proteins. Patterns of change were analysed by clustering using Netwalker 1.0, Pearson coefficient and significance analysis of microarrays (SAM) correction.
Cluster analysis, SAM multiplex testing and paired comparison of individual analytes identified proteins that were upregulated or downregulated during treatment with HICS. There was upregulation of the hypothalamo-pituitary-adrenal axis after HICS treatment evidenced by increases in α-MSH and ACTH in cases treated with HICS. Interestingly, significant increase in PIIINP was associated with HICS treatment and improved MRSS suggesting that this may be a marker of extracellular matrix turnover. Other relevant factors reduced in HICS-treated patients compared with controls, although not reaching statistical significance included COMP, CCL2, IL6, TIMP2, Fractalkine and TGFβ1 levels.
Our results suggest mechanisms of action for HICS, including upregulation of α-MSH, that has been shown to be anti-fibrotic in preclinical models, and possible markers to be included in future trials targeting skin in diffuse cutaneous systemic sclerosis.
Eudract, No. 2007-003122-24. ClinTrials.gov, No. NCT00769028. Registered 7 October 2008.
KeywordsScleroderma Clinical trial Biomarker Goat serum Melanocortin
Hyperimmune caprine serum (HICS) has been reported to have beneficial effects in several disease settings with potential improvement in several neurological and inflammatory human diseases [1–5]. The mechanism of action in these conditions is poorly understood and likely multifactorial. Proposed pharmacodynamic effects of HICS include an effect on ion channel function [6–9], immunosuppression and neuroendocrine modulation through the hypothalamo-pituitary axis .
Recently reported clinical data from a phase II study in established diffuse cutaneous systemic sclerosis (dcSSc) suggest possible treatment benefit of HICS for skin over 26 weeks for active treatment compared with placebo and meaningful improvement in 50% of cases, and improvement in modified Rodnan skin score (MRSS) in an extended dataset including subjects that moved from placebo to active treatment after the first 26 weeks . Here we report the effect of treatment on multiple serum proteins and examine the potential association between changes in analytes and modified Rodnan skin score in individual study subjects.
In addition to elucidating the effect of HICS, this systematic study of biologically relevant candidate molecular markers in a cohort of established dcSSc gives new insight to a phase of the disease that is associated with substantial morbidity and mortality, but that has often been excluded from clinical trials that generally focus on early diffuse systemic sclerosis (SSc). In this way we have highlighted the potential benefit of biological intervention/immunomodulation in established SSc and also demonstrated that information about skin treatment may be derived from this population.
Study cohort and clinical outcomes
This was a placebo-controlled study of 20 patients with established diffuse cutaneous SSc, defined by disease duration of at least 36 months from first non-Raynaud’s symptom. Subjects were randomly allocated 1:1 to active treatment or placebo. The clinical and demographic features of the study cohort have been described in detail previously . Of note, three patients withdrew from the study; one in the HICS group due to cerebral infarction unrelated to study medication and two in the placebo group due to progression of disease. After 26 weeks of blinded treatment, all patients were offered open-label treatment for a further 26 weeks. The blind of original treatment allocation was maintained to 52 weeks. The flow of subjects for the study is shown in Additional file 1: Figure S1. For the primary analysis, baseline and 26-week MRSS were compared and statistical difference between the active treatment and placebo-treated cases was compared. Secondary analysis differentiated responders from non-responders defined by improvement in skin score by at least 20% of baseline and four MRSS units. Post hoc analysis of MRSS change included seven placebo cases that moved to the active treatment and three cases that continued without medication.
Serum and plasma protein analysis
Blood samples were obtained at baseline, week 0 (pre- and post-injection of medication), weeks 26 and 52. Serum and plasma samples were sent frozen to Quest Diagnostics (Valencia, CA, USA) for single-protein analysis or Quansys Biosciences (Logan, UT, USA) for multiplex analysis, as outlined below.
Single-analysis assays were used for analysis of procollagen III N-terminal propeptide (PIIINP), soluble interleukin-2 receptor (sIL-2R), cartilage oligomeric matrix protein (COMP), transforming growth factor beta 1 (TGF-β1) and von Willebrand factor (vWF). vWF samples were plasma samples and an enzyme-linked immunosorbent assay (ELISA) (Cat. No. 84793; Aushon Biosystems, Inc., Billerica, MA, USA). Soluble IL-2R samples were serum samples and an ELISA (Cat. No. EH2IL2R; Thermo Fisher Scientific, Waltham, MA, USA). TGF-β1 samples were serum samples and an ELISA (Cat. No. DB100B; R&D Systems, Inc., Minneapolis, MN, USA). PIIINP samples were serum samples and a radioimmunoassay (Cat. No. 06098; Orion Diagnostica Ltd., Espoo, Finland).
Multiplex serum analysis was performed for αMSH, ACTH, ANG2, HGF, PDGF-bb, TIMP-1, TIMP-2, VEGF, FGF basic, Eotaxin, GRO-α, MCP-1, MCP-2, RANTES, I-309, TARC, IP-10, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, IL-23, IFN-γ, TNF-α, TNF-β, IFN-α, IFN-β, Fractalkine and PARC by multiplex analysis. Samples were Quansys Biosciences by Q-Plex Array™ kits for Human Angiogenesis (No. 150251HU), Human Chemokine (No. 120251HU), and Human Cytokine (No. 110951HU). Both Fractalkine and PARC were custom developed from matched pair antibodies available from R&D Systems. The Q-Plex™ kits used in the sample testing have undergone extensive validation. Ranges for each assay were determined by dilutions determining upper ranges where high-end hook effect and apparent antibody saturation are avoided and lower ranges that are above detection limits. Antigen standard curves were performed in duplicate.
For each analyte there was an individual comparison of baseline, 26 and 52 weeks and an integrated multiplex analysis to look for clusters of change in groups of cytokines that may reflect treatment with HICS or clinical differences in MRSS occurring during the study period. Unsupervised and supervised cluster analysis was undertaken to define baseline differences or changes over 26 and 52 weeks. Differences were compared between treatment groups and also for those with subgrouping strategies at baseline, longitudinally and linked to meaningful or numerical change in clinical variables. Permutation analysis was used to compare cytokine levels between the treatment arm and placebo arm. This was processed in EXCEL (Microsoft Corp., Redmond, WA, USA) and analysed using t tests with significance analysis of microarray (SAM) correction. Normalisation of data points and hierarchical clustering were performed using Netwalker 1.0 (http://netwalkersuite.org), and heat map construction was performed using CIMminer (https://discover.nci.nih.gov/cimminer/). Scatter plots were used to explore the association between MRSS and individual analytes. Baseline and 26-week values were compared between HICS and control treatment arms in the extended dataset and changes were also examined in the subset of subjects that changed from placebo to HICS at 26 weeks and for those that received HICS over a total of 52 weeks.
Representative serum analytes (mean [SD]) for subjects receiving HICS or placebo treatment over 26 weeks
Change during study HICS
Change during study placebo
HICS versus placebo
Direction of change
26 wk versus basal p value
26 wk versus basal p value
n = 10
n = 9
n = 9
n = 10
n = 10
n = 10
FRACT (CX3CL1) ng/ml
CCL2 (MCP1) pg/ml
Clinical outcome data for modified Rodnan skin score
Serum and plasma protein analysis
The data for the 26-week placebo-controlled phase of the study for PIIINP, vWF and sIL2R have been presented previously . Here we show additional data to week 52 that confirm the earlier findings (Table 1). For the baseline measurements, there were 20 subjects, at 26 weeks there were n = 10 receiving placebo and n = 9 receiving HICS due to one patient discontinuing treatment early in the study. Fifty-two-week follow-up was on a compassionate basis with safety review and so fewer samples were available. For clarification, the total study cohort was 20 subjects. There were samples available for assay from n = 7 subjects who had moved from placebo to HICS, n = 6 subjects continuing HICS treatment and n = 2 subjects who moved from HICS to no treatment and n = 3 subjects who were in the placebo arm and chose not to receive HICS from weeks 26 to 52. The flow of patients through the study is illustrated in the schematic in Additional file 1: Figure S1.
Cluster analysis and heat maps
To provide a clearer signal of potential serum protein changes seen in the SSc subjects receiving active treatments in the placebo-controlled phase of the study, a summary heat map was generated for the placebo-controlled phase of the study. This demonstrates that some markers are shared and these may be markers of clinical change or response. In contrast the signature comparing HICS and placebo (Additional file 4: Figure S4) is more likely to be a direct effect of the HICS administration and may therefore be a better reflection of the impact of this novel therapy rather than the clinical changes occurring in MRSS, that might also provide insight into potential markers of change in skin severity that may be independent of treatment effect (Additional file 4: Figure S4). For the key analytes highlighted in Table 1 the heat maps are annotated showing that for almost all of these the change seen with HICS treatment compared with placebo was also observed in analysis based upon the responder status for MRSS.
Statistical analysis of changes in serum proteins associated with HICS treatment
We next undertook statistical analysis of the whole dataset using SAM. The strength of this methodology is that it takes account of the multiple simultaneous analyses that are performed and corrects the data for false discovery risk. From this analysis a signature of proteins up- or downregulated by HICS emerged and these proteins were then subjected to a more detailed analysis of change over the 52 weeks of the study. These proteins are shown in Table 1 together with the basal and 26-week level, fold change and significance assessed by SAM and for individual statistical analysis of the analytes.
At an individual patient level levels of some analytes, including COMP, TGFβ1, αMSH and PIIINP that showed significant correlation for baseline MRSS confirming previous published data from other independent studies. However, there was no correlation between changes in these analytes and MRSS change during the study at an individual patient level (data not shown).
In this study we demonstrate the value of including biological analyses exploring potential markers and mechanisms of treatment effect in clinical trials of novel potential therapeutics, such as hyperimmune caprine serum . By including later stage dcSSc we highlight the feasibility of recruiting this subgroup that may have some advantages (e.g. clinical homogeneity, acceptability of requiring no other immunosuppression and acceptability of a true placebo control) compared with early-stage SSc . It is notable that there was progression in the placebo group in this study that is contrary to recent suggestions that later stage dcSSc is likely to be in a regressive stage . A potential explanation for this is that immunosuppressive treatments were withdrawn in the study subjects and this may have led to more active skin disease.
Simultaneous measurement of multiple analytes is a powerful approach that has been applied in cross-sectional analyses but few longitudinal studies [15, 16]. We identify a molecular signature of HICS response in SSc and correlate with potential treatment effect that includes proteins that increase with HICS treatment compared with untreated cases, especially PIIINP, αMSH and ACTH, and also proteins that showed trends of reduction in the HICS-treated subjects. Our findings suggest potential for development of composite serum biomarkers for HICS response in SSc and could inform development of a more generic composite serum biomarker for SSc [17–19]. This would complement other markers that include serum variables and tests of other available markers such as the enhanced liver fibrosis (ELF) test  and confirm the feasibility of this approach. Our study has the particular strength of having longitudinal sampling and simultaneous assessment of MRSS. The changes observed for HICS treatment are notable in the context of falling MRSS. For PIIINP, the changes are unexpected in that there is increase in the context of improved MRSS both in the overall treatment cohort for HICS and also in the responder analysis (Additional file 3: Figure S3). This suggests that PIIINP may be a marker of extracellular matrix (ECM) remodeling as well as fibrotic burden as discussed below. Other proteins showing significant change include markers of activation (αMSH and ACTH) with highly significant increase at 26 weeks suggesting sustained upregulation of the hypothalamic pituitary axis in response to HICS. COMP has previously been suggested as a marker of skin score in scleroderma and so a trend for reduction in HICS-treated cases but not controls in notable.
Several distinct possible effects of HICS that have previously been suggested are highly relevant to the study. First, HICS is likely to have an immunomodulatory effect. This may reflect the presence of proteins and cytokines that affect immune function , including polyclonal immunoglobulin that has previously been suggested to be beneficial for some aspects of SSc. A recent retrospective single-centre observational study of 30 patients with refractive dcSSc receiving intravenous immunoglobulin (IVIG) showed significant reduction in MRSS at 24 months, indicating that it may be an effective adjunctive treatment . To date, only one randomised double-blind trial has been completed. In this trial, a single 5-day course of IVIG did not show significant improvement but a retreatment with a second course showed an improvement in skin score . Other recent reports suggest benefits in gastrointestinal symptoms and myositis in SSc patients receiving IVIG [24, 25].
One of the most compelling potential anti-fibrotic mechanisms for HICS is through stimulation of the hypothalmo-pituitary axis. It is notable that there is considerable evidence that stimulation of MSH pathways may benefit preclinical animal models of fibrosis and in vitro studies with human tissue [26–28]. For example, Bohm et al. described that human dermal fibroblasts express the MC1 receptor (MC1R) that binds α-MSH with high affinity and they found that α-MSH suppressed TGF-β-induced collagen synthesis in vitro [29, 30]. Furthermore, the authors used a bleomycin mouse model to investigate the effects of α-MSH on skin fibrosis and found that simultaneous administration of α-MSH with bleomycin suppressed the effects of bleomycin on HDF. ACTH was also found to have similar suppressive effects. α-MSH exerts its effects via a cAMP-driven pathway and not via Smad 2/3. α-MSH upregulates superoxide dismutase 2 and hemeoxygenase 1, which is protective against the effects of bleomycin on reactive oxygen species. They also confirmed the presence of POMC and the MC1R in affected skin from patients with SSc and dermal fibroblasts strongly expressed both POMC and MC1R . In a recent study MC1-signalling-deficient mice were susceptible to bleomycin-induced fibrosis, whereas wild-type animals were not .
Strengths of this work include prospective definition of clinical phenotype, collection within the framework of a double-blind clinical trial and standardised assay methodology. Longitudinal sampling of candidate makers and placebo control data over a clinically meaningful duration allows more robust interpretation than in cross-sectional studies. There are also some limitations such as the relatively small number of study subjects in a heterogeneous disease so that subgroups may be distributed unevenly between the treatment groups. The need to use different platforms for some single-factor assays may be a limitation compared with using the same methods for all analyses. In addition, conclusions may only apply to late-stage diffuse cutaneous SSc. It would be useful in future work to extend this and include other stages and subsets of SSc such as limited cutaneous disease (lcSSc) or early diffuse SSc. The effect of previous immunosuppression cannot be reliably explored in this study but interplay between HICS and other more conventional immunomodulatory approaches that are in use as treatment for SSc or its complications could be addressed in future work.
These findings provide important information about future potential for SSc molecular markers. They provide important insight into the potential biological effects of HICS in SSc and in other medical indications. The data are hypothesis generating and suggest potential future therapeutic avenues to explore such as the effect of HICS in a larger and more diverse clinical cohort, and possible development of new composite serum markers that reflect changes in MRSS in clinical trials across the disease spectrum.
cartilage oligomeric matrix protein
Diffuse cutaneous systemic sclerosis
Enzyme-linked immunosorbent assay
Hyperimmune caprine serum
Modified Rodnan skin score
Procollagen III N-terminal propeptide
Significance analysis of microarray
Transforming growth factor beta 1
von Willebrand factor
The clinical trial was sponsored and funded by Daval International. Additional biomarker analysis and interpretation was supported by a research grant from EULAR as part of their Orphan Diseases Programme.
Daval International sponsored this clinical trial and funded this work. Analysis and additional work was supported by European League Against Rheumatism (EULAR) through the Orphan Diseases Programme.
Availability of data and materials
NQ designed the study, collected, analysed and interpreted clinical and laboratory data, drafted the manuscript, critically revised the manuscript and approved the final version. KC analysed and interpreted multiplex cytokine data, drafted the manuscript, critically revised the manuscript and approved the final version. BY conceived and designed the study, analysed and interpreted data, critically revised the manuscript and approved the final version. JV analysed and interpreted statistical aspects of the data, critically revised the manuscript and approved the final version. DM designed the study, analysed and interpreted data, critically revised the manuscript and approved the final version. SH designed the study, analysed and interpreted data, critically revised the manuscript and approved the final version. CD conceived and designed the study, collected, analysed and interpreted data, drafted the manuscript, critically revised the manuscript and approved the final version.
DM and JV are employees of Daval International. SH is a consultant for Daval International. No other competing interests.
Consent for publication
Ethics approval and consent to participate
All subjects gave informed consent and the study was approved by the Hampstead Research Ethics committee.
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- Mackenzie R, Kiernan M, McKenzie D, Youl BD. Hyperimmune goat serum for amyotrophic lateral sclerosis. J Clin Neurosci. 2006;13(10):1033–6.View ArticlePubMedGoogle Scholar
- Mackenzie RA. Follow-up study of hyper-immune goat serum (Aimspro) for amyotrophic lateral sclerosis (ALS). J Clin Neurosci. 2009;16(11):1508–9.View ArticlePubMedGoogle Scholar
- Youl BD, Ginsberg L. Goat serum product AIMSPRO® shows promise as an effective treatment in CIDP. London: BSCN meeting, National Hospital; 2004.Google Scholar
- Youl BD, Crum J. Clinical improvement in Krabbe’s disease case treated with hyperimmune goat serum product AIMSPRO®. J Neurol Sci. 2005;238:S110.Google Scholar
- Youl BD, Angus-Leppan H, Hussein N, Brooman I, Fitzsimons RB. Rapid and sustained response to hyperimmune goat serum product in a patient with Myaesthenia Gravis. J Neurol Sci. 2005;238:S177.Google Scholar
- Moore CEG, Hannan R, McIntosh D. In vivo, human peripheral nerve strength duration time constant changes with AIMSPRO® implicate altered sodium channel function as a putative mechanism of action. J Neurol Sci. 2005;238:S238.Google Scholar
- Kiernan MC, Burke D, Bostock H. Nerve excitability measures: biophysical basis and use in investigation of peripheral nerve disease use in investigation of peripheral nerve disease. In: Dyck PJ, Thomas PK, editors. Peripheral Neuropathy. 4th ed. Philadelphia: Elsevier Saunders; 2005. p. 113–29.View ArticleGoogle Scholar
- Burke G, Cavey A, Matthews P, Palace J. The evaluation of a novel ‘goat serum’ (AIMSPRO®) in multiple sclerosis. J Neurol Neurosurg Psychiatr. 2005;76:1326.Google Scholar
- Youl BD, White SDT, McIntosh D, Cadogan M, Dalgleish AG, Ginsberg L. Hyperimmune serum reverses conduction block in demyelinated human optic nerve and peripheral nerve fibres. J Neurol Neurosurg Psychiatr. 2004;76:615.Google Scholar
- Youl BD, Orrell R. Goat serum product AIMSPRO® produces sustained improvement in muscle power in a patient with fascioscapulohumeral dystrophy. J Neurol Sci. 2005;238:S169.Google Scholar
- Quillinan NP, McIntosh D, Vernes J, Haq S, Denton CP. Treatment of diffuse systemic sclerosis with hyperimmune caprine serum (AIMSPRO): a phase II double-blind placebo-controlled trial. Ann Rheum Dis. 2014;73(1):56–61.View ArticlePubMedGoogle Scholar
- Chung L, Denton CP, Distler O, Furst DE, Khanna D, Merkel PA. Clinical trial design in scleroderma: where are we and where do we go next? Clin Exp Rheumatol. 2012;30(2 Suppl 71):S97–102.PubMedGoogle Scholar
- Khanna D, Furst DE, Allanore Y, Bae S, Bodukam V, Clements PJ, et al. Twenty-two points to consider for clinical trials in systemic sclerosis, based on EULAR standards. Rheumatology (Oxford). 2015;54(1):144–51.View ArticleGoogle Scholar
- Maurer B, Graf N, Michel BA, Müller-Ladner U, Czirják L, Denton CP, Tyndall A, Metzig C, Lanius V, Khanna D, Distler O. Prediction of worsening of skin fibrosis in patients with diffuse cutaneous systemic sclerosis using the EUSTAR database. Ann Rheum Dis. 2015;74:1124–31.View ArticlePubMedGoogle Scholar
- Beirne P, Pantelidis P, Charles P, Wells AU, Abraham DJ, Denton CP, et al. Multiplex immune serum biomarker profiling in sarcoidosis and systemic sclerosis. Eur Respir J. 2009;34(6):1376–82.View ArticlePubMedGoogle Scholar
- Vettori S, Cuomo G, Iudici M, D’Abrosca V, Giacco V, Barra G, et al. Early systemic sclerosis: serum profiling of factors involved in endothelial, T-cell, and fibroblast interplay is marked by elevated interleukin-33 levels. J Clin Immunol. 2014;34(6):663–8.View ArticlePubMedGoogle Scholar
- Pendergrass SA, Hayes E, Farina G, Lemaire R, Farber HW, Whitfield ML, et al. Limited systemic sclerosis patients with pulmonary arterial hypertension show biomarkers of inflammation and vascular injury. PLoS One. 2010;5(8):e12106.View ArticlePubMedPubMed CentralGoogle Scholar
- Rice LM, Ziemek J, Stratton EA, McLaughlin SR, Padilla CM, Mathes AL, et al. A longitudinal biomarker for the extent of skin disease in patients with diffuse cutaneous systemic sclerosis. Arthritis Rheumatol. 2015;67(11):3004–15.View ArticlePubMedGoogle Scholar
- Chakravarty EF, Martyanov V, Fiorentino D, Wood TA, Haddon DJ, Jarrell JA, et al. Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis. Arthritis Res Ther. 2015;17:159.View ArticlePubMedPubMed CentralGoogle Scholar
- Abignano G, Cuomo G, Buch MH, Rosenberg WM, Valentini G, Emery P, et al. The enhanced liver fibrosis test: a clinical grade, validated serum test, biomarker of overall fibrosis in systemic sclerosis. Ann Rheum Dis. 2014;73(2):420–7.View ArticlePubMedGoogle Scholar
- Thacker JD, Brown MA, Rest RF, Purohit M, Sassi-Gaha S, Artlett CM. 1-Peptidyl-2-arachidonoyl-3-stearoyl-sn-glyceride: an immunologically active lipopeptide from goat serum (Capra hircus) is an endogenous damage-associated molecular pattern. J Nat Prod. 2009;72(11):1993–9.View ArticlePubMedGoogle Scholar
- Poelman CL, Hummers LK, Wigley FM, Anderson C, Boin F, Shah AA. Intravenous immunoglobulin may be an effective therapy for refractory, active diffuse cutaneous systemic sclerosis. J Rheumatol. 2015;42(2):236–42.View ArticlePubMedGoogle Scholar
- Takehara K, Ihn H, Sato S. A randomized, double-blind, placebo-controlled trial: intravenous immunoglobulin treatment in patients with diffuse cutaneous systemic sclerosis. Clin Exp Rheumatol. 2013;31(2 Suppl 76):151–6.PubMedGoogle Scholar
- Raja J, Nihtyanova SI, Murray CD, Denton CP, Ong VH. Sustained benefit from intravenous immunoglobulin therapy for gastrointestinal involvement in systemic sclerosis. Rheumatology (Oxford). 2016;55(1):115–9.View ArticleGoogle Scholar
- Clark KE, Etomi O, Denton CP, Ong VH, Murray CD. Intravenous immunogobulin therapy for severe gastrointestinal involvement in systemic sclerosis. Clin Exp Rheumatol. 2015;33(4 Suppl 91):S168–70.PubMedGoogle Scholar
- Lee TH, Jawan B, Chou WY, Lu CN, Wu CL, Kuo HM, et al. Alpha-melanocyte-stimulating hormone gene therapy reverses carbon tetrachloride induced liver fibrosis in mice. J Gene Med. 2006;8(6):764–72.View ArticlePubMedGoogle Scholar
- Zhang Z, Ma J, Yao K, Yin J. Alpha-melanocyte stimulating hormone suppresses the proliferation of human tenon’s capsule fibroblast proliferation induced by transforming growth factor beta 1. Mol Biol (Mosk). 2012;46(4):628–33.Google Scholar
- Luo LF, Shi Y, Zhou Q, Xu SZ, Lei TC. Insufficient expression of the melanocortin-1 receptor by human dermal fibroblasts contributes to excess collagen synthesis in keloid scars. Exp Dermatol. 2013;22(11):764–6.View ArticlePubMedGoogle Scholar
- Bohm M, Raghunath M, Sunderkotter C, Schiller M, Stander S, Brzoska T, et al. Collagen metabolism is a novel target of the neuropeptide alpha-melanocyte-stimulating hormone. J Biol Chem. 2004;279(8):6959–66.View ArticlePubMedGoogle Scholar
- Bohm M, Eickelmann M, Li Z, Schneider SW, Oji V, Diederichs S, et al. Detection of functionally active melanocortin receptors and evidence for an immunoregulatory activity of alpha-melanocyte-stimulating hormone in human dermal papilla cells. Endocrinology. 2005;146(11):4635–46.View ArticlePubMedGoogle Scholar
- Kokot A, Sindrilaru A, Schiller M, Sunderkotter C, Kerkhoff C, Eckes B, et al. alpha-melanocyte-stimulating hormone suppresses bleomycin-induced collagen synthesis and reduces tissue fibrosis in a mouse model of scleroderma: melanocortin peptides as a novel treatment strategy for scleroderma? Arthritis Rheum. 2009;60(2):592–603.View ArticlePubMedGoogle Scholar
- Bohm M, Stegemann A. Bleomycin-induced fibrosis in MC1 signalling-deficient C57BL/6 J-Mc1r(e/e) mice further supports a modulating role for melanocortins in collagen synthesis of the skin. Exp Dermatol. 2014;23(6):431–3.View ArticlePubMedGoogle Scholar