This cross-sectional study compared data and findings of patients with SD/DU to those with CSU and healthy controls (three groups of participants), specifically, data on the intensity of perceived psychological stress (using validated questionnaires), as well as on salivary cortisol and IL-1β levels and the clinical characteristics of the disease. Furthermore, this study aimed to examine their interrelations and correlations, as well as their association with demographic data (gender, age, concomitant conditions, triggers). The purpose of the investigation was to determine whether patients with SD/DU experience higher levels of psychological stress, and whether they exhibit changed IL-1β levels compared to CSU patients and healthy controls.

A total of 100 participants were included in the study: 34 patients with SD/DU, 33 patients with CSU, and 33 healthy controls (those with SD/DU associated with CSU were excluded). The patients were triaged during routine examinations at the Allergy and Clinical Immunology Outpatient Clinic, Department of Dermatovenereology, University Hospital Center “Sestre Milosrdnice”, where the study was conducted. For all enrolled patients, disease severity and clinical parameters were evaluated. Each participant provided written informed consent, and the study received approval from the Ethics Committee of the abovementioned hospital (July 2023; number of protocol: 251-29-11-23-07). Inclusion criteria for the SD/DU and CSU groups were age ≥ 18 years; presence of wheals and/or angioedema persisting for more than 6 weeks, in accordance with current guidelines for the diagnosis of chronic urticaria (2). Patients with isolated angioedema were excluded. Exclusion criteria for all participants included use of systemic corticosteroids, antineoplastic agents, psychoactive medications, immunosuppressive/immunomodulatory drugs, or vaccines within one month prior to inclusion; use of oral contraceptives; for women, being in the follicular phase of the menstrual cycle; use of topical corticosteroids within 7 days prior to sampling; presence of systemic inflammatory or autoimmune disease, history of malignancy, diagnosed psychiatric disorder, or oral diseases; and smoking (the known influence of nicotine on salivary cortisol levels)! (24). Healthy controls were matched by age and sex to the patient groups and met the following criteria: age over 18 years, absence of dermatological, inflammatory, or autoimmune disease. For all participants, saliva samples were collected and analyzed for IL-1β and cortisol levels using the same standardized procedure.

Clinical disease parameters were evaluated using validated instruments: Urticaria Control Test (UCT) for evaluating disease control (25); Urticaria Activity Score (UAS7) for assessing disease activity in CSU patients (2); and the Perceived Stress Scale (PSS) for measuring the intensity of psychological stress (it measures stress during the period of the last 1 month) (26, 27). In addition, demographic and clinical data were collected, including age, sex, and disease duration. Further data were also analyzed, regarding comorbidities (none; allergic rhinosinusitis, asthma, pollinosis, oral allergy syndrome, concomitant type I allergies, etc.); and self-reported identified triggers.

In patients with SD/DU, we used the FricTest to assess the current severity of the dermographism, i.e., the skin’s reaction as an indicator of response to provocation testing (FricTest^® 4.0, Moxie, Germany), which was applied on the volar forearm of the patients (28–30) (Figure 1). The test was performed while patients were at rest, not taking any additional medication for urticaria, and having discontinued second-generation AHs for at least 3 days prior to testing. All procedures were conducted by the same investigator, at the same time of day between 07:00 and 09:00 a.m. We measured the wheal diameter that appeared 10 minutes after provocation. The observed responses in each patient were recorded as negative (−) or positive, scored from + to ++++, i.e. from 1–4 according to the threshold value that elicited the reaction (wheal and/or itching). Thus, we recorded the results for each patient: the higher the FricTest score, the greater the activity of SD/DU (29).

Morning saliva samples were collected between 07:00 and 09:00 a.m., corresponding to the circadian peak of cortisol, and were used for the determination of cortisol and IL-1β concentrations (following completion of the PSS questionnaire) (26, 31, 32). Saliva collection was performed by passive drool, avoiding sampling within 90 minutes after a major meal or tooth brushing, and 12 hours after alcohol consumption. To minimize the effects of low pH and bacterial growth, participants rinsed their mouths with water 10 minutes prior to collection. They then allowed saliva to accumulate in the mouth for approximately 20 minutes, and subsequently tilted their heads forward to guide the pooled saliva into a collection vial (Salivette^®, Sarstedt, Nümbrecht, Germany), yielding approximately 2.0–2.5 mL of saliva. Samples were transported immediately after collection to the Department of Clinical Chemistry and frozen at −20 °C within 4 hours. On the day of cortisol and IL-1β determination by Salivary ELISA Kits (Salimetrics^®, PA, USA). The samples were thawed, vortexed, and centrifuged at 1500 x g for 15 minutes.

The normality of data distribution was tested using the Kolmogorov–Smirnov test. Non-normally distributed data were log-transformed to approximate normality. For normally distributed variables, comparisons among the three groups were performed using analysis of variance with the Student–Newman–Keuls post hoc test. For non-normally distributed variables, the Kruskal–Wallis test was applied, followed by the Mann–Whitney U test with Bonferroni correction. Effect size was calculated using the formula r = Z/√N for the Mann–Whitney U test. The correlation between variables was analyzed using Spearman’s rank correlation coefficient. The magnitude of effect size and correlation strength was interpreted according to Cohen’s criteria where r = 0.25–0.3 indicates a small effect/low correlation, r = 0.3–0.5 is moderate, r = 0.5–0.7 is large, and r > 0.7 = a very large correlation. Frequencies were compared using Fisher’s exact test. All analyses were performed using IBM SPSS Statistics, version 22.0 (IBM Corp., Armonk, NY, USA).

A total of 100 participants were included in the study: 34 patients with SD/DU, 33 patients with CSU, and 33 healthy controls, aged 19–64 years (median 38; interquartile range 31–48), of whom 78% were women. Age and sex distribution did not differ significantly between the three groups. Duration of SD/DU varied between 9 and 180 months, with median 36 (interquartile range 80-60). An analysis of salivary factors and perceived stress levels showed that there were no significant differences in salivary cortisol and IL-1β concentrations, or perceived stress levels (PSS scores) between the groups (Figures 2–4;Table 1). When assessing disease control, the CSU group demonstrated significantly higher disease control compared to the SD/DU group, with a moderate effect size (p = 0.001; r = 0.393) (Figure 5). Disease duration in SD/DU patients was negatively linearly related to salivary cortisol (r=-0.369; p=0.032), but not to IL-1β. No significant correlations were observed between salivary cortisol, IL-1β, perceived stress, and age in the overall sample (including both patients and controls). Values of salivary cortisol, IL-1β, and perceived stress were not associated with patient age but did show sex-related differences, with small effect sizes. Higher IL-1β concentrations were observed in male patients compared to female patients (median 687.6 vs. 408.9 pg/mL; p=0.029; r=0.218). Similarly, salivary cortisol levels (log-transformed values) were higher in males than in females (median 1.1 vs. 0.9; p=0.028; r=0.220). Conversely, perceived stress was higher in female patients compared to males (median 16 vs. 12; p = 0.006; r = 0.273). For CSU patients, a significant positive correlation was found between disease severity (UAS7) and PSS (moderate, linear and positive correlation, indicating that higher perceived stress/PSS was associated with greater disease severity/UAS7). Regarding disease control and activity of two patient groups in relation to sex and age, no significant associations were found—both UCT (both diseases) and UAS7 results (CSU) were independent of sex and age.

While dermographism activity/severity (on a scale from 1 to 4) did not correlate with most examined factors (UCT, cortisol, or IL-1), they did correlate with PSS (r=0.347; p=0.045), with a positive, linear, and moderate correlation (Figure 6). As perceived stress increased, so did dermographism activity levels. However, PSS did not differ significantly between dermographism activity levels (Figure 7). Table 2 shows a comparison of data on concomitant/associated diseases and reported triggers for urticaria between the patient groups. SD/DSU patients who reported stress as a trigger had significantly higher disease control/UCT (8.5 vs. 6; p=0.042; r=0.348). Also, CSU patients who reported stress as a trigger had a higher disease severity/UAS7 (median 24 vs. 10.5; p=0.035; r=-0.362, a moderate effect size). Also, SD/DU patients with concomitant allergies had lower cortisol, with a moderate effect size (median log 1.03 vs. 0.80 nmol/L; p=0.032; r=-0.368). SD/DU lasted longer in those with concomitant conditions (60 vs. 24 months; p=0.017; r=-0.410), particularly in those with concomitant allergies (median 48 vs. 18 months; p=0.002; r=-0.51; a large effect size), especially when allergic to aeroallergens (median 54 vs. 24 months; p=0.011; r=-0.485; a moderate effect size).

The limitations of this study (which was the first simultaneously analyzed psychological stress, immunological factors and clinical features in SD/DU patients) are the relatively small number of patients and the lack of other potentially valuable data like stress duration (considering medical data that long-lasting stress can reduce morning cortisol level), as well as the possibility that salivary IL-1β is a potentially more useful indicator of oral conditions, and not reliable indicator of chronic urticaria. Since our results indicate high perceived stress in SD/DU patients and since the duration of their illness was negatively linearly related to salivary cortisol (supporting chronic stress), reducing psychological stress could help improve the condition of SD/DU. This highlights the need for further studies of biomarkers of SD/DU and associated stress and for larger, more comprehensive studies.