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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: J Pediatr Gastroenterol Nutr. 2021 Sep 1;73(3):367–375. doi: 10.1097/MPG.0000000000003181

Associations Between Subclass Profile of IgG Response to Gluten and the Gastrointestinal and Motor Symptoms in Children with Cerebral Palsy

Reidun Stenberg 1,2,3,#, Melanie Uhde 1,2,#, Mary Ajamian 1,4, Peter H Green 1,2, Anna Myleus 5, Armin Alaedini 1,2,4,7,§
PMCID: PMC8380641  NIHMSID: NIHMS1706760  PMID: 34231978

Abstract

Objective.

Gastrointestinal problems are often seen in children with cerebral palsy, although the etiology and underlying mechanisms are not fully understood. Recent data point to significantly elevated levels of IgG antibody to dietary gluten in cerebral palsy independent of celiac disease, a gluten-mediated autoimmune enteropathy. We aimed to further characterize this antibody response by examining its subclass distribution and target reactivity in the context of relevant patient symptom profile.

Methods.

In a previous study, we investigated whether an elevated frequency of celiac disease might partially explain the observed weight and height impairment among children with CP. Serum IgG antibody to gluten was investigated for subclass distribution, pattern of reactivity towards target proteins, and relationship with gastrointestinal symptoms and motor function.

Results.

The anti-gluten IgG antibody response in the cerebral palsy cohort was comprised of all four subclasses. However, in comparison with celiac disease, IgG1, IgG2, and IgG3 subclasses were significantly lower, whereas the IgG4 response was significantly higher in cerebral palsy. Within the cohort of cerebral palsy patients, levels of anti-gluten IgG1, IgG3, and IgG4 were greater in those with gastrointestinal symptoms, and the IgG3 subclass antibody correlated inversely with gross motor function. The anti-gluten IgG antibodies targeted a broad range of gliadin and glutenin proteins.

Conclusion.

These findings reveal an anti-gluten IgG subclass distribution in cerebral palsy that is significantly different from that in celiac disease. Furthermore, the observed association between IgG subclass and symptom profile is suggestive of a relationship between the immune response and disease pathophysiology that may indicate a role for defects in gut immune and barrier function in cerebral palsy.

WHAT IS KNOWN

  • While gastrointestinal problems are common in children with cerebral palsy (CP), contributing mechanisms and relevance to motor function have not been delineated.

  • Previous data point to an enhanced IgG response to gluten in CP, although whether and how this antibody response may differ from that in celiac disease are not known.

WHAT IS NEW

  • The subclass profile of elevated IgG response to gluten in CP is significantly different in comparison with celiac disease and is associated with greater gastrointestinal and motor symptoms.

  • The data are suggestive of a relationship between the immune response and disease pathophysiology that may indicate a role for the evolution of B cell immunity and defects in gut immune and barrier function in CP.

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Keywords: cerebral palsy, celiac disease, gluten sensitivity, immune activation, B cell, immunoglobulin, antibody subclass, gastrointestinal symptoms, motor function

INTRODUCTION

Cerebral palsy (CP) is the most common, severe, and economically costly physical disability in children, with a prevalence of 1.5 to more than 4 per 1,000 live births or children of a specific age range (1, 2). In addition to the compromised motor control as a central feature, CP is associated with seizures, behavioral deficits, and gastrointestinal (GI) symptoms, as well as hearing, vision, and cognitive disturbances (3, 4). Malnutrition and failure to thrive are often observed in CP, but underlying causes have been inadequately explored and are likely to be multifactorial and include reduced calorie and nutrient intake due to oro-motor deficits and muscle spasticity, endocrine abnormalities, GI problems, and potential environmental factors (5).

Celiac disease (CD) is an autoimmune enteropathy that is also associated with malnutrition, failure to thrive, and impaired growth in pediatric patients (6). The disease is triggered by ingestion of gluten from wheat and related cereals, resulting in intestinal inflammation, villous damage, and crypt hyperplasia in genetically susceptible individuals. Celiac disease is strongly associated with genes for the specific class II human leukocyte antigens (HLA) DQ2 and DQ8, which are involved in presenting specific immunogenic peptides of gluten proteins to CD4+ T cells in the small intestine (7). The disease is also closely associated with elevated immune response to gluten proteins and to the transglutaminase 2 (TG2) autoantigen (8).

In a previous study, we investigated whether an elevated frequency of celiac disease might partially explain the observed weight and height impairment among children with CP (9). The study demonstrated significantly higher levels of IgG antibody to gluten compared with matched controls. The increased IgG response to gluten was associated with a greater degree of disability, but no connection was found between the elevated antibody response to gluten and the presence of celiac disease among children with CP. A follow-up study demonstrated the presence of more modestly elevated levels of IgG antibodies to other food antigens in CP children (10).

Despite their elevated levels, the expression of antibody reactivity to gluten in CP is likely to be mechanistically distinct from celiac disease. We have shown that, unlike celiac disease, the occurrence of these antibodies in CP is not related to the expression of HLA-DQ2/DQ8 or the anti-TG2 antibody response (9, 10). In children with celiac disease, the systemic IgG antibody response to gluten has been known to comprise primarily of the IgG1 and IgG3 subclasses (11, 12). Furthermore, celiac disease is associated with immune reactivity towards a broad range of gliadin and glutenin protein sequences in gluten (13, 14). Whether and how the subclass distribution and target protein reactivity of the IgG antibody response to gluten in CP may differ from those in celiac disease have not been examined. In this study, we aimed to further investigate the observed immune reactivity to gluten in CP by examining the IgG subclass distribution and by performing an exploratory analysis of the target specificity of IgG reactivity towards individual gluten protein subgroups, including gliadins and glutenins. Potential relationships between these antibodies and the presence of GI symptoms and motor function deficits are also explored.

METHODS

Patients and Controls.

The study included serum from 70 children with CP, recruited between 2003 and 2005 in Sweden. The diagnosis and subtyping of CP were made by a pediatric neurologist according to previously described definitions and criteria (15). Assessment of the level of gross motor function in individuals with CP was made according to the Gross Motor Function Classification System (GMFCS), which describes five levels from I (most able) to V (most limited) (16). The total disability load was assessed from a combination of the CP classification and GMFCS score. None of the CP patients in this cohort had celiac disease or elevated levels of IgE-antibody to wheat or gluten.

The study also included serum from 85 Swedish children with celiac disease, recruited between 1992 and 1995 (17). All cases of celiac disease were positive for IgA anti-transglutaminase 2 (TG2) autoantibody, biopsy-proven, and diagnosed according to established criteria (18). Children in the CP and celiac disease cohorts were on a gluten-containing diet at the time of serum collection.

In addition, the study included 30 healthy controls on non-restricted diets, recruited at child health care centers and schools in Sweden, as described previously (19).

All samples were collected with written informed consent under relevant institutional review board-approved protocols at Umeå, Örebro, and Uppsala in Sweden. Serum specimens were kept at −80 °C to maintain stability. This study was approved by the Institutional Review Board of Columbia University Medical Center.

Gluten proteins.

Preparation of the gluten antigen for the immunoassays was as previously described (20). One hundred mg of the U.S. hard red spring wheat Triticum aestivum Butte 86 flour was suspended in 1 mL of 40% ethanol and mixed for 30 min at room temperature. The suspension was centrifuged at 10,000 g for 15 min. The supernatant was removed, added to 1.9 mL of 1.5 M NaCl, and stored at 4 °C overnight. The precipitate was removed by centrifugation, rinsed with H2O, and dissolved in 0.2 mL of 0.1 M glacial acetic acid. The extracted gluten solution was lyophilized and stored at −20 °C.

Assays.

Serum levels of total IgG reactivity to gluten and individual IgG subclass reactivities to gluten were measured separately by enzyme-linked immunosorbent assay (ELISA) according to the previously described protocol (21, 22), with some modifications as follows. A 2 mg/mL stock solution of the gluten antigen was prepared in 70% ethanol. Wells of 96-well Maxisorp round-bottom polystyrene plates (Nunc) were coated with 50 µL/well of a 0.01 mg/mL solution of protein in 0.1 M carbonate buffer (pH 9.6) or left uncoated to serve as controls. After incubation at 37°C for 1 h, all wells were washed and blocked by incubation with 1% bovine serum albumin (BSA) in PBS containing 0.05% Tween-20 (PBST) for 1.5 h at room temperature. Serum samples were diluted at 1:300, added at 50 µL/well in duplicates, and incubated for 1 h. Each plate contained a positive control sample with a high level of IgG or relevant IgG subclass reactivity to gluten, as determined in a preliminary screen. After washing, the wells were incubated with HRP-conjugated anti-human IgG1 (Life Technologies), IgG2 (Life Technologies), IgG3 (Life Technologies), or IgG4 (Southern Biotech) secondary antibodies for 50 min. The plates were washed and 50 μL of developing solution, containing 27 mM citric acid, 50 mM Na2HPO4, 5.5 mM o-phenylenediamine, and 0.01% H2O2 (pH 5), was added to each well. After incubating the plates at room temperature for 20 min, absorbance was measured at 450 nm. All serum samples were tested in duplicate. Absorbance values were corrected for non-specific binding by subtraction of the mean absorbance of the associated BSA-coated control wells. The corrected values were first normalized according to the mean value of the positive control duplicate on each plate. The mean antibody level for the healthy control cohort was then set as 1.0 AU and all other results were normalized to this value. The cutoff value for anti-gluten IgG positivity was assigned as two standard deviations above the mean for the healthy control group.

Assessment of IgG antibody reactivity towards the various gluten protein groups was examined by immunoblotting as we have previously described (13, 23), with some modifications. Extracted gluten protein (0.66 μg protein/lane) was dissolved in sample buffer, heated for 10 min at 75°C, and separated by SDS-PAGE using NuPAGE 10% bis-tris gels (Invitrogen). Protein transfer onto nitrocellulose membranes was carried out with the iBlot Dry Blotting System (Life Technologies). The membranes were incubated for 1 h in a blocking solution made of 5% milk and 0.5% bovine serum albumin in a solution of Tris-buffered saline containing 0.05% Tween-20 (TBST). Incubation with patient serum (1:4000) in dilution buffer containing 10% blocking solution and 10% fetal bovine serum in TBST was done for 1 h. Serum samples from representative CP patients with elevated IgG antibody reactivity to gluten, as well as celiac disease and healthy controls representing positive and negative controls, were included. HRP-conjugated anti-human IgG (GE Healthcare) was used as secondary antibody. Detection of bound antibodies was by the ECL system (Millipore) and the FluorChem M imaging system (ProteinSimple).

Data analysis.

Group differences were analyzed by the Kruskal-Wallis one-way analysis of variance with post-hoc testing. Correction for multiple comparisons was done using Dunn’s statistical hypothesis testing and the multiplicity-adjusted P values are reported for each comparison. Adjustment for covariate effect (age and sex) was carried out for all comparisons by analysis of covariance, using the general linear model. Correlation analysis was performed using Spearman’s r. All P values were 2-sided, and differences were considered statistically significant at P<0.05. Statistical analyses were performed with Prism, version 8 (GraphPad) and Minitab, version 19 (Minitab) software.

RESULTS

Patients and controls.

The study cohorts’ clinical and demographic characteristics are shown in Table 1.

Table 1.

Demographic and clinical characteristics of study cohorts.

Subject group Number of subjects Mean age—years [SD] Female sex—no. (%) Celiac disease-associated HLA DQ2 and/or DQ8—no. (%) Celiac disease-associated anti-TG2 antibody positivity—no. (%)
CP 70 9.6 [4.3] 27 (38) 35 (50) 1 (1.4)
Celiac disease 85 6.6 [3.6] 51 (60) 85 (100) 85 (100)
Healthy 30 9.8 [3.6] 11 (37) - 0
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IgG subclass distribution.

Of the 85 celiac disease and 70 CP patients, serum samples from 73 patients with celiac disease and 26 individuals with CP exhibited anti-gluten IgG antibody reactivity above the cutoff for positivity. Levels of total anti-gluten IgG antibody reactivity were not significantly different between the antibody-positive celiac disease and CP groups. Sera from the anti-gluten IgG-positive patients were further analyzed for anti-gluten IgG subclass distribution.

The anti-gluten IgG antibody response in the celiac disease group was comprised mainly of IgG3 and IgG1 subclasses, and to a lesser extent, IgG2 subclass, as these antibodies were significantly increased in comparison to both the healthy control group (P<0.0001 for all three subclasses) and CP cohort (P<0.0001, P=003, and P=0.0003, respectively) (Fig. 1A–C). The celiac disease group exhibited no increase in the IgG4 subclass antibody response to gluten (Fig 1D).

Figure 1.

Figure 1.

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Levels of IgG subclass antibody to wheat gluten. Serum levels of (A) IgG1, (B) IgG2, (C) IgG3, and (D) IgG4 antibody to the extracted gluten antigen in cohorts of unaffected controls and anti-gluten total IgG-positive celiac disease and CP patients, as determined by ELISA. Gray bars indicate the median for each cohort.

While the CP patients also had significantly elevated levels of IgG1, IgG2, and IgG3 antibodies compared to the healthy control group (P<0.0001, P=0005, and P=0.002, respectively), the levels of these antibodies were significantly lower than in celiac disease patients (P=0.003, P=0003, and P<0.0001, respectively) (Fig. 1A–C). In contrast with the celiac disease group, the IgG4 subclass also contributed to the total IgG anti-gluten antibody response in the CP cohort, with levels being significantly higher than in healthy controls and celiac disease patients (P<0.0001 for both comparisons) (Fig. 1D).

There was not an association between HLA genotype and any subclass IgG reactivity to gluten for the CP cohort.

Gastrointestinal (GI) symptoms.

Medical histories were available for 60 of the 70 children with CP. Among them, 29/60 (48.3%) were experiencing persistent GI symptoms, including 26 with constipation, 8 with gastroesophageal reflux, and 3 with chronic loose stools or diarrhea.

Patients with the above GI symptoms exhibited significantly greater levels of total anti-gluten IgG antibody than those without (P=0.002) (Fig. 2A). Similarly, CP patients who were positive for anti-gluten IgG antibody reactivity (according to the criteria described in Methods) had more GI symptoms than the antibody-negative CP patients (P=0.003). In addition, CP patients with constipation had significantly higher levels of IgG antibody to gluten when compared to those without constipation (P=0.016) (Fig. 2B). Among the IgG-positive CP patients, those with constipation had modestly higher levels of IgG1 (p = 0.034), IgG3 (p = 0.027), and IgG4 (p = 0.031) response to gluten in comparison to those without constipation (Fig. 2C–E). In addition, a significantly greater proportion of patients with constipation was positive for IgG4 antibody to gluten (10 of 13) than patients without constipation (1 of 9) (P=0.008). No other significant differences were found with regard to other subclasses or GI symptoms.

Figure 2.

Figure 2.

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Levels of anti-gluten IgG in the context of GI symptoms in CP patients. A) CP patients with one or more GI symptoms had higher levels of IgG antibody to gluten than those with symptoms. B) CP patients with constipation had higher levels of IgG to gluten than those without constipation. C-E) Anti-gluten total IgG-positive CP patients with constipation had higher levels of IgG1 (C), IG3 (D), and IgG4 (E) than anti-gluten total IgG-positive patients without constipation. Gray bars indicate the median for each cohort.

Gross motor function.

GMFCS scores were available for 63 of the 70 children with CP. There was a significant association between the presence of GI symptoms (driven primarily by constipation) and reduced gross motor function (P<0.0001) (Fig. 3A). Patients with the greatest levels of gross motor function deficit (GMFCS score=3–5) had significantly higher levels of total IgG antibody reactivity to gluten when compared with patients with the lowest degrees of deficit (GMFCS score =1–2) (P=0.008) (Fig. 3B). Similarly, CP patients who were positive for anti-gluten IgG antibody reactivity had significantly higher GMFCS scores than the antibody-negative CP patients (P=0.007). Among the anti-gluten IgG-positive CP patients, those in the high deficit (more disabled) group (GMFCS score=3–5) had significantly greater levels of IgG3 response to gluten in comparison with those in the low deficit group (GMFCS score=1–2) (P=0.004) (Fig. 3C). Additionally, there was a significant positive correlation between GMFCS score and IgG3 antibody reactivity to gluten in CP patients (r=0.529, P=0.007) (Fig. 3D).

Figure 3.

Figure 3.

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Motor function in relation to GI symptoms and anti-gluten IgG antibody response. A) Patients with GI symptoms had significantly higher GMFCS scores. B) Patients with the greatest levels of gross motor function deficit (GMFCS score=3–5) had significantly higher levels of total IgG antibody reactivity to gluten in comparison with patients with the lowest degrees of deficit (GMFCS score =1–2). C) Anti-gluten total IgG-positive CP patients in the high deficit group had significantly greater levels of IgG3 response to gluten in comparison with those in the low deficit group. D) GMFCS score and IgG3 antibody reactivity to gluten correlated significantly in anti-gluten total IgG-positive CP patients (r = 0.529, p = 0.007). Gray bars indicate the median for each cohort.

Target protein group specificity of IgG antibody response to gluten.

Subsequent to measurement of antibody levels by ELISA, serum specimens from patients identified as having elevated levels of anti-gluten total IgG were further characterized for antibody reactivity towards electrophoretically separated gluten proteins. As can be seen in Fig. 4A, the extracted gluten fraction used in these analyses included the various gliadin and glutenin protein groups. IgG antibody reactivities towards the separated gluten proteins for the CP patients with the most clearly visible binding patterns on Western blot are shown in Fig. 4D–H, in conjunction with healthy (Fig. 4B) and celiac disease (Fig. 4C) controls. The molecular specificity of the elevated IgG antibody reactivity to the separated gluten proteins in CP patients was found to be heterogeneous, targeting a range of α-, γ-, and ω-gliadins, as well as high- and low-molecular weight glutenins (Fig. 4). No single pattern of IgG binding to specific gluten proteins could be discerned.

Figure 4.

Figure 4.

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Pattern of IgG antibody reactivity towards gluten proteins in CP patients and controls. A) SDS-PAGE pattern of separation of gluten proteins extracted from the Butte 86 wheat cultivar. B-H) Western blot patterns of binding of serum IgG antibodies from representative controls (B-C) and ELISA-positive CP patients (D-H) to electrophoretically separated gluten proteins from panel A. Molecular weight markers are indicated on the right (kDa).

DISCUSSION

Our previous studies demonstrated significantly elevated levels of IgG antibody response to dietary gluten in children with CP (9, 10). However, whether and how these antibodies may differ from those found in celiac disease, the quintessential presentation of immunologic sensitivity to gluten, were unclear. The results of this study demonstrate a clear contrast in the distribution of IgG subclass reactivity towards gluten between CP and celiac disease. While the elevated IgG reactivity to gluten in both celiac disease and CP patients included IgG1, IgG2, and IgG3, the levels of the three antibody subclasses were significantly lower in CP in comparison with celiac disease. In clear contrast, levels of IgG4 anti-gluten antibody reactivity were significantly elevated in the CP cohort in comparison with the celiac disease group. In conjunction with the absence of an association between IgG anti-gluten antibodies and the HLA-DQ2/DQ8 genes or antibody response to TG2, the data are further indicative of a mechanism of immune response to gluten in CP that is distinct from celiac disease.

A number of studies have examined the molecular specificity and epitope reactivity of B and T cell responses towards gluten in patients with celiac disease. The immune response to gluten in celiac disease is triggered by and targets a broad range of gluten proteins, including various gliadins and glutenins (13, 14, 23–25). Many of the B- and T-cell immunogenic gluten sequences in celiac disease have been identified (26, 27). The data in this study indicate that, similar to celiac disease, the IgG antibody response to gluten in CP targets a broad spectrum of gluten proteins, including α-, γ-, and ω-gliadins, as well as high- and low-molecular weight glutenin subunits. Despite this, the fact that the antibody response to gluten in CP is independent of HLA-DQ2/DQ8 molecules is suggestive of significant differences in the target epitope specificity of the anti-gluten immune response between CP and celiac disease, which need further in-depth examination in future studies.

Subclass switching of the IgG response towards an antigen evolves in a 1-way direction from IgG3 to IgG1, IgG2, and IgG4 over time (28). Among the IgG subclasses, IgG1 and IgG3 antibodies are the most potent activators of complement and are also highly efficient at binding a wide range of FcγRs on immune cells. Specifically, the IgG1 and IgG3 subclass antibodies to gluten, which are prominent in celiac disease, have been shown to have a strong complement activating capacity (12, 29). The prominence of these earlier stage subclasses in celiac disease is suggestive of repeated activation of gluten-specific naïve B cells rather than of memory cells in response to gluten exposure, despite the chronic nature of the disease. It is worth noting that while the IgG1 and IgG3 antibodies to gluten have the potential to contribute to localized inflammation by fixing complement and other effector functions through interaction with tissue-bound gluten (30) or cross-reactivity with autoantigens (31, 32), a direct pathogenic impact remains to be proven.

The IgG4 subclass antibody contains unique structural properties that distinguish it from other immunoglobulin isotypes and IgG subclasses. IgG4 binds weakly to Fc receptors and to complement (33, 34), and due to the poor stability of binding between its heavy chains and the resulting ease of Fab-arm exchange, it is not efficient at crosslinking of antigens or forming immune complexes (35). IgG4 has also been shown to induce an anti-inflammatory M2-like macrophage phenotype through inhibition of interferon gamma (IFNγ) signaling (36). Considering these properties, it has been suggested that IgG4 can regulate the immune response and limit inflammation through competition with other antibodies (33, 34, 37). It is therefore conceivable that the enhanced IgG4 response to gluten in CP, possibly driven by repeated exposure to the antigen due to defects in the intestinal barrier that may facilitate greater antigen translocation, is indicative of a mechanism directed at limiting the potential pathologic impact of the IgG1 and IgG3 subclasses.

Elevated levels of IgG4 antibodies are found in a number of conditions, including allergies and hypersensitivities, infections, malignancies, autoimmunity, and IgG4-related disease (38). In allergies, IgG4 responses are protective against severe hypersensitivity and anaphylaxis triggered by the allergen, mediated in part through competition with IgE and complement-binding IgG subclasses. In addition, expansion of IgG4-switched B cells occurs in allergen immunotherapy and correlates with allergen tolerance. On the other hand, IgG4 antibodies in helminthic infections and malignancies can interfere with the effective clearance of the parasite or tumor cells. Furthermore, in certain autoimmune and immune-mediated diseases, IgG4 antibodies directed at autoantigens have been found to be pathogenic in passive transfer experiments (38). Similarly, in IgG4-related disease, an immune-mediated fibroinflammatory condition that can affect multiple organs, the enhanced IgG4 B cell responses have been found to be directed towards self-antigens. However, the potential role of the IgG4 in the pathogenesis of IgG4-related disease remains unclear and continues to be a subject of debate (39). Considering that these antibodies are directed at autoantigens, their mechanism of development and action is not likely to be related to the IgG4 response to dietary gluten in CP patients.

Although there is increasing acknowledgement of immune abnormalities in cerebral palsy, it is often unclear whether they are reflective of a pathologic process or a secondary consequence (40, 41). Here, we found that CP patients with GI symptoms (primarily constipation) exhibit significantly higher levels of IgG reactivity to gluten, driven by IgG1, IgG3, and IgG4 subclasses. In addition, a greater proportion of patients with constipation were positive for the IgG4 antibody to gluten than those without constipation. Data from a previous study point to an association between chronic constipation and increased indicators of intestinal permeability (42). Increased intestinal permeability has been shown to be associated with microbial translocation, leading to enhanced systemic immune reactivity to microbial and dietary antigens, including gluten (22, 43). It is conceivable that repeated systemic antigen exposure due to the constipation-associated intestinal permeability would lead to an enhanced B cell switch towards the more advanced IgG subclasses, culminating in the elevated IgG4 response. However, it should be noted that a study of patients with irritable bowel syndrome found no association between constipation and elevated levels of IgG4 to dietary and microbial antigens (44). Clearly, there is a need for more in depth examination of the potential connection between constipation, enhanced antibody response to luminal antigens, and IgG subclass switching through further studies.

The results of this study also indicate that patients with more limited gross motor function have increased IgG antibody reactivity to gluten, and that among the IgG subclasses, levels of IgG3 correlate with severity of gross motor disability. The observed correlation between levels of anti-gluten IgG3—the less advanced IgG subclass response with strong inflammatory effector functions—and reduced motor function may lend further credence to other lines of evidence for increased systemic inflammation in CP. However, it is important to note that these data do not by themselves provide evidence in support of a role for antibodies in the pathophysiology of CP. Further mechanistic studies are needed to gain a more nuanced understanding of the relevance of the mucosal and systemic B cell and other immune responses in the context of CP pathophysiology.

In the absence of celiac disease or wheat allergy, it is speculated that the underlying mechanisms for the enhanced antibody reactivity to gluten in CP may share some overlap with a condition referred to as non-celiac wheat or gluten sensitivity (NCGS). NCGS patients experience a range of intestinal and extra-intestinal symptoms in response to ingestion of gluten-containing cereals, in the absence of celiac disease or IgE-mediated wheat allergy (45). The condition is associated with IgG reactivity to gluten at levels in par with celiac disease (22). There is now evidence for a biological basis in NCGS that includes extensive systemic innate and adaptive immune activation in conjunction with a compromised intestinal epithelial barrier (22). NCGS patients exhibit elevated levels of antibody to gluten and microbial antigens, along with a robust innate immune response to translocated microbial products (22). It has also been shown that the anti-gluten IgG antibody response in NCGS is characterized by a significantly lower contribution of IgG1 and IgG3 subclasses in comparison to celiac disease that is compensated by higher IgG4 and IgG2 subclass antibody reactivities (46). The findings have been interpreted as being suggestive of a sustained primary B cell response to gluten in celiac disease despite the condition’s chronicity (similar to this study’s pediatric celiac disease cohort), and a more advanced and tolerogenic immune response to gluten in NCGS (46). Considering the similarities between NCGS and a significant proportion of CP patients in the IgG subclass profile and relationship with the HLA-DQ2/DQ8 genetic background, future work in the context of CP should include examination of intestinal barrier function, microbial translocation, and systemic immune activation in relation to GI and extraintestinal manifestations.

In summary, the results of this study extend earlier data on the observed immune reactivity to gluten in children with cerebral palsy by providing antibody subclass information that is further indicative of mechanisms distinct from those in celiac disease and may point to pathways directed at inhibition of inflammation in CP. Furthermore, the intriguing associations between specific IgG subclasses and patient GI and motor symptoms are suggestive of a link between immunologic response to antigens and disease pathophysiology in CP that could implicate gastrointestinal system dysfunction. This may include defects in the mucosal immune response and enteric nervous system, damage to the gut epithelial barrier, and microbial dysbiosis. As such, further in-depth investigation of the relevance of gastrointestinal system defects and immune abnormalities to CP pathophysiology is warranted.

ACKNOWLEDGMENT

We thank Dr. Dan Hellberg from the Center for Clinical Research at Uppsala University in Falun, Sweden for providing access to serum samples from healthy controls. We are also grateful to Dr. Donald D. Kasarda at the Western Regional Research Center of the U.S. Department of Agriculture for providing us with the Butte 86 wheat flour used for the gluten extractions.

Funding Sources:

This study was supported by the National Center for Advancing Translational Sciences (to A.A.), Nexttobe AB (to A.A.), and Promobilia (to R.S.). The funding agencies had no role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the manuscript.

Abbreviations:

CP

cerebral palsy

ELISA

enzyme-linked immunosorbent assay

FcγR

Fc-gamma receptor

GI

gastrointestinal

GMFCS

Gross Motor Function Classification System

HLA

human leukocyte antigen

NCGS

non-celiac gluten/wheat sensitivity

TG2

transglutaminase 2

Footnotes

Competing financial interests: The authors declare no competing interests.

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