Evaluation of serum VIP and aCGRP during pulmonary exacerbation in cystic fibrosis: A longitudinal pilot study of patients undergoing antibiotic therapy

**Background:** Cystic fibrosis (CF) is the most common fatal genetic disease among Caucasians, characterized by multisystem inflammation and progressive lung destruction. Pulmonary exacerbations are clinically important events that can lead to permanent loss of lung function. Objective monitoring of treatment response is difficult in children who cannot perform pulmonary function testing (FEV1). The identification of easily measurable blood biomarkers that predict response to therapy is therefore a high priority. Vasoactive intestinal peptide (VIP) and alpha calcitonin gene-related peptide (aCGRP) are neuropeptides with immunomodulatory, bronchodilatory, and mucus-regulating functions in the airways. This study aimed to investigate serum levels of VIP and aCGRP in CF pediatric patients during pulmonary exacerbation and after antibiotic therapy, and to explore associations with clinicopathological parameters.

**Methods:** Twenty-one CF patients were prospectively recruited at the onset of pulmonary exacerbation (defined by at least 4 of 12 Fuchs criteria) from three hospitals in Jordan between July 2018 and August 2019. Exclusion criteria included transplant recipients and patients on chronic immunosuppressive therapy. All patients received 2 weeks of antibiotic therapy plus airway clearance therapies. Peripheral blood (5 ml) was collected at three time points: hospital admission before antibiotics (VIP1, aCGRP1), 3 days post-antibiotic (VIP2, aCGRP2), and at discharge (2 weeks post-antibiotic; VIP3, aCGRP3). Serum VIP and aCGRP levels were measured using human ELISA kits (CLOUD-CLONE CORP, USA). Clinical variables recorded included age, gender, BMI, CF-related diabetes, liver disease, sputum microbiology, and antibiotic type. Statistical analysis used repeated measures MANOVA with backward selection; log transformation was applied to normalize VIP data. P ≤ 0.05 was considered significant.

**Key Results:** The mean age was 8.71 years (SEM 1.02); 14 patients (66.7%) were male. Mean BMI z-score was -2.09 (SEM 0.61). Nine patients (42.9%) had comorbidities; 2 (9.5%) had diabetes mellitus; 3 (14.3%) had liver disease. Nine patients (42.9%) were positive for Pseudomonas aeruginosa, and 15 (71.4%) were positive for Staphylococcus aureus (1 MRSA). Antibiotics prescribed included ciprofloxacin (47.6%), azithromycin ± amoxicillin/clavulanic acid (38.1%), cefixime (9.5%), and gentamicin (4.8%). For VIP, overall least square (LS) means did not change significantly over time (p > 0.05). However, significant between-subjects effects were found for diabetes mellitus (p = 0.026), other comorbidities (p = 0.013), and type of antibiotic (p = 0.019). Patients with diabetes or comorbidities had higher VIP levels; levels were highest with ciprofloxacin and lowest with cefixime. For aCGRP, overall LS means changed significantly over time (p = 0.005): LS mean started at 364.5, increased to 350.2 at 3 days, then decreased slightly to 349.8 at 2 weeks. Significant between-subject effects were found for type of medication (p = 0.012) and positive S. aureus infection (within-subject effect, p = 0.046). Patients with positive S. aureus had lower aCGRP levels than those without. Forest plot analysis of univariate differences (VIP2-VIP1, VIP3-VIP1, aCGRP2-aCGRP1, aCGRP3-aCGRP1) showed all 95% confidence intervals contained zero, indicating no significant pairwise differences between time points for individual subgroups.

**Clinical Implications:** This is the first study to investigate serum VIP and aCGRP levels in CF patients during pulmonary exacerbation and after antibiotic therapy. The significant change in aCGRP levels over time suggests it may serve as a candidate biomarker for monitoring response to exacerbation treatment, particularly in children where pulmonary function testing is not feasible. The association of aCGRP with S. aureus infection aligns with prior evidence of its antimicrobial activity. The lack of significant change in VIP levels may reflect its complex regulatory role or the small sample size. The association of VIP with diabetes mellitus is consistent with its known insulinotropic and anti-inflammatory effects. However, due to the small sample size (n=21), lack of a healthy control group, non-standardized antibiotic regimens across sites, and absence of FEV1 data, these findings are preliminary. Larger cohort studies with standardized protocols and mechanistic investigations are needed to validate these biomarkers for clinical use in CF management.