Study design and subjects
We conducted this prospective interventional study enrolling 30 subjects, male and female, between age 18 and 50, with IBS, at 6 general practitioners offices in Genova, Italy. All the subjects had to meet Rome-IV criteria for the diagnosis of IBS, consisting in recurrent abdominal pain at least 1 day/wk, on average, in the last 3 months, with symptoms beginning at least in the last 6 months, associated with two or more of the following alterations: (1) related to defecation; (2) associated with a change in frequency of stool; and (3) associated with a change in appearance of stools [21]. Major exclusion criteria were: known inflammatory bowel disease (e.g. Crohn, Ulcerative Colitis), alcohol abuse, eating behavior disorders, diabetes, coeliac disease, chronic therapy with drugs known to alter intestinal motility or adsorption (e.g. thyroxine, PPIs).
Anthropometric measurements (weight, height, circumference at waist, abdomen and hips) and an IBS symptom assessment (Irritable Bowel Syndrome Symptom Severity Score, IBS-SSS) [22] was obtained by the general practitioner at enrollment, after 3 weeks and after 6 weeks (end of study, EOS). At enrollment and after 6 weeks we obtained a blood sample via finger puncture for the measurement of BAFF, PAF and food-specific IgG. After enrollment and after each visit performed by the general practitioner (week 3 and EOS), a telephone consultation with a specialized nutritionist was offered to explain the dietary intervention and to evaluate compliance.
Dietary intervention
The proposed nutritional pattern agreed with the Healthy Eating Plate proposed by Harvard School of Public Health [23]. The daily total energy intake was distributed across three main meals (breakfast, lunch, and dinner). Based on the food-specific IgG measurement and relative distribution, a personalized food profile was created for each subject identifying 1 to 3 relevant food groups/nutritional clusters [24]. Subjects were then instructed to avoid the foods highlighted in their personal food profile in certain days of the week, and to assume them only in 7 of the 21 meals of the week (two full days and another meal of choice). In this way each subject was instructed to restrict the food groups (highlighted in their own personal results) that were more frequently consumed in their own usual diet (i.e. gluten, dairy, nickel), forcing the consumption of aliments from different food groups and thus increasing food variability. The adherence to this plan, achieving at least a whole day of avoidance of the food groups in their personal profile was the determining factor in IBS symptom and food-related IgG reduction.
No calorie restriction was imposed in the diet. Compliance was assessed via the combined analysis of a 3-day food diary compiled by each subject and 48-h food recollection obtained during the telephone consultations with a specialized nutritionist at week 3 and week 6. Given the instructions to avoid the food groups in almost 5 days of the week (with assumption permitted only in two full days plus another meal of choice), subjects were considered non-compliant (NC) if they failed to achieve at least one full day of abstinence from the food groups in the combined assessment.
Laboratory studies
B-cell Activating factor (BAFF), and Human Platelet Activating Factor (PAF) were measured via commercial ELISA kits (Human BAFF/BLyS/TNFSF13B Immunoassay Quantikine® ELISA, Catalog Number PDBLYS0B, R&D Systems Inc, Minneapolis, MN, USA; Human Platelet Activating Factor ELISA Kit, Catalog Number E-EL-H2199, Elabscience Houston, TX, USA, respectively) using the Biomek 4000 ELISA microplate liquid reagent dispensing automation tool (Beckman Coulter, Brea, CA, USA) and the EL405LS ELISA microplate automated washing system (BioTek Instruments, Winooski, VT, USA). The absorbance of each well was read at a wavelength of 450 nm with a Multiskan FC plate reader (Thermo Scientific, Waltham, MA, USA). The average zero standard optical density was subtracted from all absorbances, and a standard curve was generated by using a four-parameter logistic (4-PL) curve fit. The concentration in the test sample was calculated through interpolation along the standard curve by multiplying the result by the dilution factor.
Serum IgG responses to food antigens is evaluated by sensitive and specific ELISA (enzyme-linked immunosorbent assay) test using the Automatic Workstation Biomek i7 (Beckman Coulter, Brea, CA, USA). Food antigens and reference antigen for standards and controls are bound to the plate. Diluted patient serum or standards are pipetted into the wells of the microtiter plate. A binding between the IgG antibodies of the serum and the immobilized antigens takes place. After one hour incubation at 37 °C, the plate is rinsed with diluted wash solution in order to remove unbound material. Then Goat anti-human-IgG-HRP conjugate (Catalog Number 62-8420, Thermo Fisher Scientific, Rockford, IL, USA) is added and incubated for 30 min at 37 °C. After a further automatic washing step, the substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) solution (Surmodics IVD Inc., Eden Praire, MN, USA) is pipetted and incubated for 20 min at 37 °C, inducing the development of a blue colour in the wells. The colour development is modified by the addition of a stop solution (sulfuric acid 2 N). The resulting yellow colour is measured spectrophotometrically at the wavelength of 450 nm. The concentration of the IgG antibodies is directly proportional to the intensity of the colour. The standards of the IgG ELISA are commercially available human IgG defined and expressed in IU/mL (cat. n. I4506, Sigma Aldrich, St. Louis, MO, USA). This results in an exact and reproducible quantitative evaluation. Consequently for a given patient follow-up controls become possible. For a quantitative evaluation the absorbances of the standards are graphically drawn against their concentrations. From the resulting reference curve the concentration values for each patient sample can then be extracted in relation to their absorbances. The lower limit of detection is 0.1 IU/mL with the intra- and inter-assay reproducibility of 4.9% and 6.9%, respectively.
Statistical analysis
Statistical analysis of the data was performed using GraphPad Prism 8 for macOS (GraphPad Software, San Diego, CA, USA. Version 8.3.0 (328), 16 October 2019). The data is presented as median and range. The medians were compared using the Mann–Whitney test. A p value < 0.05 was used as the limit of statistical significance.