Background & Aims

Fibromyalgia (FM) is a complex chronic pain disorder affecting between 0.2% and 6.6% of people worldwide, especially middle-aged women (1-2). This condition is defined by persistent widespread pain, chronic fatigue, sleep problems, cognitive issues and comorbidities like migraines, major depression, rheumatologic conditions (3). FM etiopathogenesis is not clearly understood and it seems to involve genetic and environmental factors (psychological and physical stressors) and biological elements (4). There are no specific laboratory tests and the lack of FM biomarkers makes the diagnosis and therapy arduous and sometimes useless. We identified Mu opioid receptor positive (Mu+) B cell percentage as a candidate marker, named Mu Lympho Marker (MLM), for chronic pain FM and osteoarthritis patients (5-6). Here we investigated whereas MLM remains stable over time, in the same patients after 2 years. Moreover we studied Mu opioid receptor cellular localization, through confocal microscopy.

Methods

This is an observational, longitudinal diagnostic study. All FM participants previously enrolled in the first trial (5) were called back for follow-up. Patients currently using opioids were excluded to avoid potential interactions with Mu opioid receptor modulation. On the day of enrollment, clinical data were collected, and pain scores were reported using the 11-point Numerical Rating Scale (NRS), ranging from 0 for “no pain” to 10 for “the worst possible pain” (7). Peripheral blood samples were collected to analyze the percentage of Mu+ B cells expression, using flow cytometry with a Mu opioid receptor-specific antibody, on an LSR Fortessa X20 flow cytometer (BD). Peripheral Blood Mononuclear Cells (PBMC) were collected for immunofluorescence analysis to visualize Mu cellular localization on B cells using an Eclipse 80i Nikon Fluorescence Microscope.

Results

The totality of the participants were female, ranked between 18 and 65 years old, mean age 54±8.8. Twenty-eight (28) of the 59 patients already enrolled were FM patients, forty-three (43) were pain-free healthy people. Patients’ pain intensity ranked between moderate (4-6) to severe (7-10) pain (mean: 7.6±0,2 ) and no significant differences, in NRS score, were detected between the first and follow-up study. Flow cytometry results showed intra-group homogeneity and the percentage of Mu+ B cells was statistically lower in FM patients than in Pain-Free subjects, as demonstrated in the first study. Such results remained stable over time, with no significant differences. Immunoflorescence imaging showed a cytoplasmic localization of Mu receptor, corresponding to an internalization of the receptor in FM patients, differently from pain free control subjects.

Conclusions

A good biomarker should be fast detectable, economically sustainable and stable in firm conditions. Here we propose MLM as a good FM biomarker because of its easy and cheap detection and, above all, its stability reflected in intra-group homogeneity and in the difference in Mu+ B cell percentages between FM patients and pain-free subjects, which remain steady over time. Moreover, patients on prolonged opioid therapy experience tolerance, which is associated with the cellular internalization of opioid receptors (8). We recruited opioid-free patients to bypass any potential pharmacodynamic influence on Mu receptors. As a result, further investigations are necessary to ascertain whether the observed internalization of Mu receptors in FM patients is attributed to the potential overexpression of endogenous peptides or involves other yet unknown mechanisms. Overall, a precise identification of pain patients will pave the way for customized pharmacological and rehabilitation plans.

References

1.Nicholas, M., Vlaeyen, J. W., Rief, W., Barke, A., Aziz, Q., Benoliel, R., … & Treede, R. D. (2019). The IASP classification of chronic pain for ICD-11: chronic primary pain. Pain, 160(1), 28-37
2.Marques, A. P., Santo, A. D. S. D. E., Berssaneti, A. A., Matsutani, L. A., & Yuan, S. L. K. (2017). Prevalence of fibromyalgia: literature review update. Revista brasileira de reumatologia, 57, 356-363.
3.Varallo, G., Scarpina, F., Arnison, T., Giusti, E. M., Tenti, M., Rapelli, G., … & Castelnuovo, G. (2023). Suicidal ideation in female individuals with fibromyalgia and comorbid obesity: prevalence and association with clinical, pain-related, and psychological factors. Pain medicine, pnad139.
4.Häuser,W.; Sarzi-Puttini, P.; Fitzcharles, M.A. Fibromyalgia syndrome: Under-, over- and misdiagnosis. Clin. Exp. Rheumatol.2019, 37, 90–97.
5.Raffaeli W, Malafoglia V, Bonci A, Tenti M, Ilari S, Gremigni P, Iannuccelli C, Gioia C, Di Franco M, Mollace V, Vitiello L, Tomino C, Muscoli C. Identification of MOR-Positive B Cell as Possible Innovative Biomarker (Mu Lympho-Marker) for Chronic Pain Diagnosis in Patients with Fibromyalgia and Osteoarthritis Diseases. Int J Mol Sci. 2020 Feb 22;21(4):1499. doi: 10.3390/ijms21041499. PMID: 32098316; PMCID: PMC7073128.
6.Malafoglia V, Ilari S, Gioia C, Vitiello L, Tenti M, Iannuccelli C, Cristiani CM, Garofalo C, Passacatini LC, Viglietto G, Scavalli AS, Tomino C, Mollace V, Raffaeli W, Di Franco M, Muscoli C. An Observational Study on Chronic Pain Biomarkers in Fibromyalgia and Osteoarthritis Patients: Which Role for Mu Opioid Receptor’s Expression on NK Cells? Biomedicines. 2023 Mar 17;11(3):931. doi: 10.3390/biomedicines11030931. PMID: 36979910; PMCID: PMC10046119.
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8.Cox BM, Crowder AT. Receptor domains regulating mu opioid receptor uncoupling and internalization: relevance to opioid tolerance. Mol Pharmacol. 2004 Mar;65(3):492-5. doi: 10.1124/mol.65.3.492. PMID: 14978226.

Acknowledgements:
GR-2021-12375174
PON03PE_00078_1, PON03PE_00078_2
SG-2021-12375551

Presenting Author

Valentina Malafoglia

Poster Authors

Valentina Malafoglia

PhD

IRCCS SAN RAFFAELE- Rome

Lead Author

Sara Ilari

IRCSS SAN RAFFAELE, ROME

Lead Author

Tenti Michael

PhD

Fondazione ISAL, Institute for Research on Pain

Lead Author

Leonardo Lupacchini

IRCCS San Raffaele Roma

Lead Author

Laura Vitiello

IRCCS San Raffaele Roma

Lead Author

Stefania Proietti

Uniroma5

Lead Author

Lucia Carmela Passacatini

IRCCS San Raffaele

Lead Author

Carlo Tomino

IRCCS San Raffaele

Lead Author

Vincenzo Mollace

Department of Health Sciences; University "Magna Graecia" of Catanzaro, Institute of Research for F

Lead Author

Cristina Iannuccelli

Rheumatology Unit, Department of Internal Clinical, Anesthesiologic and Cardiovascular Sciences, Sap

Lead Author

Chiara Gioia

Rheumatology Unit, Department of Internal Clinical, Anesthesiologic and Cardiovascular Sciences, Sap

Lead Author

Manuela Di Franco

Rheumatology Unit, Department of Internal Clinical, Anesthesiologic and Cardiovascular Sciences, Sap

Lead Author

William Raffaeli

Fondazione ISAL, Institute for Research on Pain

Lead Author

Carolina Muscoli

Department of Health Sciences; University "Magna Graecia" of Catanzaro, Institute of Research for F

Lead Author

Topics

  • Assessment and Diagnosis