Mucosal Penetration and Clearance of Gluten and Milk Antigens in Eosinophilic Oesophagitis

Anupama Ravi; Eric V. Marietta; Jeffrey A. Alexander; Kathryn Peterson; Crystal Lavey; Debra M. Geno; Joseph A. Murray; David A. Katzka


Aliment Pharmacol Ther. 2021;53(3):410-417. 

In This Article


Patient Characteristics

Thirty-two patients with EoE and 10 controls were enrolled in this study (Table 1 and Table S1). Twenty patients had active EoE. For the EoE patients, the age range was 20–67 (mean 42 ± 12 years) and 19 were women. For controls, the age range was 25 to 83 (mean 50 ± 21 years) and was not different than EoE patients (P = 0.1750). Six of the controls were women. Twenty-two patients had active treatment at the time of biopsy including three patients on topical steroids monotherapy (2 active), 16 patients on PPI monotherapy (nine with persistent active disease therefore PPI failure) and two patients on both PPI and topical steroids (one active). Of the 10 patients off therapy, eight had active EoE. One of these patients was on mepolizumab for severe asthma. A flow chart of the patient analysis is given in the Figure 1. Staining was selectively performed in some patients based on tissue availability (one patient with inactive EoE).

Figure 1.

Flow chart of patient analysis

Presence of Food Antigen Staining

Gliadin, casein and whey were detected in oesophageal biopsies from patients with EoE (Figures 2 and 3). Overall, in patients, gluten, casein and whey were detected by positive staining in 17/32 (53.1%), 21/32 (65.6%) and 30/32 (92.0%) of patients respectively. In active EoE, 14/20(70.0%), 14/20 (70%) and 18/20 (90.0%) patients had detectable gliadin, casein and whey respectively. In inactive EoE, 3/12(25.0%), 5/12(41.6%) and 10/11(90.9%) patients with EoE had detectable gliadin, casein and whey, respectively, after elimination from diet. There was a significant difference in the presence of gliadin in active vs. inactive EoE patients (P < 0.005) but the differences for casein and whey were non-significant (P = 0.28 and P = 0.64 respectively). In control patients, no patients had detectable gliadin whereas casein and whey were detected in 2/10(20.0%) and 9/9(100%) respectively. The differences between controls and active EoE patients were significant for gliadin and casein (P = 0.001 and P = 0.026 respectively) but not for whey (P = 0.09).

Figure 2.

Immunostaining of food antigens in oesophageal mucosa in EoE and controls. Gliadin, casein, and whey staining in EoE. A, Gliadin staining in active (A1) and inactive (A2) EoE patients and controls (A3). B, Casein staining in active (B1) and inactive (B2) EoE patients and controls (B3). C, Whey staining in active (C1) and inactive (C2) EoE patients and controls (C3). Antigens are shown in red and nucleic acid in blue. ×40 magnification. Note the surface layering pattern commonly seen with whey (C3). X-axis (active, inactive, control) and Y-axis (A1-A3) gliadin, (B1-B3) Casein, (C1-C3) Whey

Figure 3.

Staining density in active EoE, inactive EoE and control patients total time after cessation of gliadin (a), casein (b) or whey (c). *P = 0.004, **P = 0.001, ***P = 0.038. Casein: active vs inactive, P = 0.36. control vs inactive, P = 0.29. Whey: active vs inactive, P = 0.55, control vs active P = 0.11

Presence of Food Antigens After Timed Avoidance

This was a differential response to the three food antigens penetration when comparing active and inactive EoE patients (Figure 4). The gliadin staining density was greater in active compared to inactive disease at ≤24 hours after exposure, P = 0.05. In contrast, no differences were detected when comparing active and inactive patients for casein and whey at ≤24 hours for presence and degree of staining. However, there was a greater density of staining for whey when compared to casein for all patients at ≤ 24 hours (mean 2.14 ± 0.91 and 1.07 ± 1.33, P = 0.02). Notably, 23/25 (92%) patients had detectable whey within 24 hours when compared to 16/26 (61.5%) patients with detectable casein during this time period (P = 0.009). Both casein and whey were present at this time point in more patients than gliadin (23/25, 16/26 and 15/26; P = 0.78 and 0.004 respectively).

Figure 4.

Staining density in active EoE, inactive EoE and control patients short (12–24 h) vs long (48–96 h) period after cessation of gliadin (A), casein (B) or whey (C). *P = 0.0080, **P = 0.0290

Six patients were assessed for the presence of food antigen present in oesophageal mucosa between 24 and 96 hours (Figure 4). Gliadin, casein and whey were detected in 2/6 (33.3%), 4/6 (66.6%) and 5/6 (84%) of patients. Interestingly, gliadin and casein were detected in the oesophageal mucosa for as long as 96 hours after the initiation of elimination by active EoE patients; whereas these were less likely to be detected in the inactive patients after 48 hours after eliminating gliadin and casein from their diet when compared to whey (Figure 4).

IgG4 Staining

As the presence of IgG4 in the oesophageal epithelium of patients with EoE appears to be a downstream marker of immunologic response to food antigen exposure, IgG4 was assessed for corroborating biologic significance of the antigen staining (Figures 5 and 6). IgG4 was found in patients with EoE more significantly when compared to controls where IgG4 was not detected (15/33, 45.5%) vs 0% (0/10, 0%) patients, P = 0.0074]. In patients with active EoE, IgG4 was present in 14/20 (70%) compared to being present in 1/12(8.3%) of inactive patients. This one patient with inactive EoE had grade 1 IgG4. IgG4 was present when stainable food antigen was also present (concordant in (15/15, 100%) patients).

Figure 5.

IgG4 staining in active EoE (A), inactive EoE (B), and control (C) ×40 magnification. Gliadin, IgG4, co-localisation of gliadin and IgG4 and nucleic acid are shown in red, green, yellow and blue respectively

Figure 6.

Staining for IgG4 in patients with active and inactive EoE for short and long periods after last antigen exposure. *P = 0.0097, **P = 0.0080