Malnourishment and retarded development of weight and height are common among children with cerebral palsy (CP) (1). The reasons for this are probably multifactorial but are still largely unknown. Even though nutritional problems in childhood CP are attracting increasing attention, and although these children receive support and help concerning nourishment, malnourishment remains a complex problem (2–4). One explanation could be insufficient uptake of nutrients over the mucosal barrier. Celiac disease (CD) is a common cause of poor growth in children and should be excluded when malabsorption is suspected. In a previous study, we found elevated levels of serum immunoglobulin A (IgA) and/or IgG antibodies against gliadin (IgA- and IgG-AGA) and/or transglutaminase 2 (TG2) to be a frequent finding (43%) among 90 children and young adults with CP. In most cases, the antibody levels were low or moderate, but high levels were found in some cases. All were negative regarding IgA-endomysium (EMA) antibodies, a highly specific CD marker. Furthermore, histopathological assessment of small-bowel biopsies did not disclose increased prevalence of CD in these children. Seropositive children had significantly lower weight, height, and body mass index, and were more severely disabled (5).
Recent data indicate that gluten-triggered small-bowel mucosal damage in CD develops gradually from mucosal inflammation to elongation of crypts and finally to overt villous atrophy. Minor small-bowel mucosal morphological and inflammatory changes such as increased density of intraepithelial lymphocytes (IELs) may indicate early developing CD (6). Increased numbers of CD3 + IELs is an unspecific finding, however, and only a minority of the subjects with such mucosal changes will eventually develop CD. Increased density of γδ+ IELs is considered more typical for CD and identification of these cells is helpful in borderline cases (7). Recently, the detection of TG2-targeted intestinal autoantibody deposits proved to be a powerful tool in diagnosing early developing CD without villous atrophy, showing a sensitivity and specificity of 93% (8).
It is known that serum EMA or TG2 antibodies of IgA class predict forthcoming CD in patients with normal small-bowel morphology (9). IgA class antibodies colocalizing with extracellular TG2 also can be demonstrated in the small-bowel mucosa, even when EMA and/or anti-TG2 antibodies are not detectable in serum, and is regarded as an early mucosal sign of CD (10,11). Approximately 90% of patients with CD are positive for the human leukocyte antigen (HLA) -DQ2 and most of the remaining 10% are HLA-DQ8-positive. Because these HLA types are common (20%–30%) in the general population, only absence of HLA-DQ2 and -DQ8 can be used to exclude CD (12).
The aim of the present study was to evaluate whether children with CP and seropositivity regarding AGA and/or anti-TG2 are positive for HLA-DQ2/DQ8 and may have signs of early developing CD in small-bowel mucosa despite normal mucosal findings according to routine analysis. Furthermore, serum antibodies against deamidated gliadin peptide (DGP) were determined, because these antibodies have higher specificity for classical CD than antibodies against native gliadin (13,14).
PATIENTS AND METHODS
Small-bowel biopsies from 15 of 16 children with CP were available for analysis of IgA deposits. These children had known elevated levels of IgA- and/or IgG-AGA and/or IgA- and IgG-TG2 (Table 1). Stored blood samples were used for the anti-DPG analysis and HLA typing. The group of children consisted of 6 girls (median age 17 years) and 10 boys (median age 9 years). The age range in both groups was 3 to 18 years (Table 1).
The diagnoses of CP were made by a pediatric neurologist, and a functional assessment of each child was made on the basis of the Gross Motor Function Classification System (GMFCS) (16).
Routine histology of the biopsies was classified according to the Swedish Society of Pathology (KVAST) document (http://www.svfp.se/node/3#Gastrointestinal):
1. Normal (compatible with Marsh 0)
2. Duodenal IELs, that is, mucosa with normal villi and thickness but with >30 IELs/100 epithelial cells (Marsh I–II); CD3 staining was done in most cases
3. Partial villous atrophy; the villi are reduced in length, but the length is greater than the breadth, and there is an increased number of IELs (Marsh IIIa)
4. Subtotal villous atrophy; the breadth of the villi is greater than the length (Marsh IIIb), or there is total villous atrophy (flat mucosa) with IELs (Marsh IIIc) (17,18)
None of the children had IgA deficiency (ie, IgA <0.07 g/L).
Small-bowel Mucosal Immunomorphology
The biopsies were freshly embedded in optimal cutting temperature compound (OCT, Tissue-Tec, Miles, Elkhart, IN), snap frozen in liquid nitrogen, and stored at −70°C. Staining was performed on 5-μm frozen sections. CD3 + IELs were stained with the monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA), γδ+ IELs with T-cell receptor (TCR) -γ antibody (Endogen Inc, Woburn, MA), and αβ+ IELs with monoclonal antibody β F1 (Endogen). Positive IELs were counted with a 100× flat-field light microscope objective in the surface epithelium; at least 30 fields of 1.6-mm epithelial length were counted and IEL density was expressed as cells per millimeter of epithelium as previously described (7–9).
The reference values for the number of cells per millimeter of epithelium are based on a study of 987 adults and set at 37 for CD3 + IELs, 25 for α/β T cells, and 4.3 for γ/δ T cells (7).
Small-bowel Mucosal TG2-specific IgA Deposits
The details of these procedures have been published elsewhere (8,10). Briefly, 5-μm unfixed cryosections of the small-bowel biopsies from 16 patients were stained for IgA and TG2 using fluorescein isothiocyanate–labeled rabbit anti-human IgA (Dako, Glostrup, Denmark) and examined by immunofluorescence microscopy. The IgA deposits were graded semiquantitatively from 0 to 3 according to their intensity along the basement membrane in the villous crypt area. For double labeling, sections also were stained for TG2 expression using monoclonal mouse antibodies against TG2 (CUB 7402, Neo Markers, Fremont, CA), followed by rhodamine-conjugated anti-mouse immunoglobulin antibodies (Dako). One investigator without earlier knowledge of the disease history or laboratory findings performed immunofluorescence microscopy.
Antibodies Against DGP
An enzyme-linked immunosorbent assay simultaneously detecting IgA and IgG antibodies against DGP (Quanta Lite Celiac DGP Screen, INOVA Diagnostics Inc, San Diego, CA) was used. The analyses were performed according to the manufacturer's instruction and the recommended cutoff level of 20 arbitrary units per milliliter was used.
HLA-DQB1 typing was performed at the Department of Clinical Immunology, University Hospital, Uppsala, Sweden, with polymerase chain reaction sequence-specific oligonucleotide probes using the Luminex flow bead platform (One Lamda Inc, Canoga Park, CA); ambiguities were resolved with a polymerase chain reaction sequence-specific oligonucleotide probes assay (Genovision, Vienna, Austria).
The regional ethics committee, Örebro and Uppsala, approved the study protocol.
The small-bowel mucosa from 1 child (with positive serum IgG-AGA and anti-DGP) showed elevated numbers of α/β+ and δ/γ+ lymphocytes, positive staining for CD3, and IgA colocalized with TG2. The mucosa from another child (with positive serum IgG-AGA and IgG-TG2) showed slightly elevated numbers of α/β+ IELs. A third patient (with positive serum IgG-AGA and IgG-TG2) had elevated numbers of δγ+ IELs. The biopsies from the 12 remaining children showed normal numbers of IELs, and no IgA deposits were found (Table 2).
Sera from 4 children were weakly positive for anti-DGP, one of which showed signs of early developing CD with mucosal IgA colocalized with TG2 (patient no. 10), whereas the mucosal histology was normal in the biopsies from the remaining 3 children (Table 2).
Ten of 16 children (62%) were positive for HLA-DQ2 and/or HLA-DQ8.
The child (no. 10) with IgA colocalized with mucosal TG2, elevated numbers of α/β+ and γ/δ+ IELs, HLA-DQ2, and positive CD3 staining (Fig. 1) also had a slightly elevated serum level of anti-DGP. She experienced constipation and had a low weight (−1, 5 SD) and height (−3, 0 SD). The CP diagnosis is tetraplegia with the functional class GMFCS V. Slightly elevated levels of IgA- and IgG-TG2 as well as IgG-AGA were previously recorded (5).
The child (no. 12) with slightly elevated numbers of α/β+ IELs and positive HLA-DQ8 had slightly elevated levels of IgG-AGA, but not IgA-AGA or anti-DGP. The CP diagnosis is diplegia with the functional class GMFCS II. He had low weight and height (−1 SD, respectively), but no gastrointestinal problems were reported.
Another child (no. 4) was positive for γ/δ+ IELs and HLA-DQ2. This child had known high levels of IgG-AGA and slightly elevated levels of IgG-TG2. The CP diagnosis was diplegia with functional class GMFCS V, and there was no report of gastrointestinal problems. The weight and height was low (−1, 5 SD and 0, 5 SD, respectively) (Tables 1 and 2).
Even though the prevalence of CD does not seem to be increased in children and young adults with CP, the diagnosis should be excluded in patients with malnourishment and nutritional problems. Malnourishment in early childhood is known to have negative effects on the central nervous system and cause cognitive dysfunction (19). Hence, it is of great importance to find treatable causes and prevent further deterioration of the cerebral function in these children. In a previous study we reported that elevated serum levels of CD-related markers (excluding IgA-EMA) is a frequent finding in children with CP, without increased prevalence of CD according to routine small-bowel histology (5). The major finding was elevated serum levels of IgG-AGA, which are of low diagnostic value for CD; however, a substantial number (30%) of these patients also had slightly elevated levels of antibodies against TG2, considered a greatly specific and sensitive marker for CD, although the serum levels were low or moderately increased in the majority of IgG class (20). The finding of mucosal IgA colocalized with TG2 makes a diagnosis of early developing CD likely in at least 1 of these children, supported by earlier observations showing that mucosal TG2-associated IgA deposits can be found in early stages of gluten intolerance despite normal villous morphology (7,10,11). In the small-bowel mucosa from another child, the number of γ/δ+ IELs was slightly increased, but no IgA deposits were found. Early developing CD can still be suspected in this case because increased density of γ/δ+ IELs is considered a relatively sensitive and specific marker of CD (21). The mucosa from a third child revealed unspecific findings of α/β+ IELs. These 3 children were HLA-DQ2 and/or HLA-DQ8 positive. Still, we do not have any explanation for our earlier findings of elevated CD-related markers in many children with CP (43%) (5). Obviously, most of the children do not have histology in line with either classical or early developing CD. Gastrointestinal motility disturbances are frequent in children with CP and may cause prolonged exposure of dietary proteins. Rectal challenge of gluten has been shown to give an inflammatory mucosal response with release of nitric oxide (22). It also is known that gliadin induces release of zonulin, a protein that induces increased intestinal permeability (23–26). The antibody production against gluten may thus not be caused by a “true” celiac inflammation, but rather a result of increased exposure of gluten to the immune system. Individuals with HLA-DQ2 and/or HLA-DQ8 may then be immunized against gluten. Further studies are warranted to elucidate whether children with CP have increased gastrointestinal permeability to other macromolecules as compared with healthy children.
The CD-related seromarkers in children with CP were predominantly of the IgG class, which have high diagnostic specificity for CD in IgA-deficient individuals (27,28). The specificity of IgG-AGA for CD is low and the importance of IgG-TG2 in the absence of IgA deficiency is not obvious.
The increased levels of CD-related markers in children with CP raise the question whether these antibodies may affect their nervous system. Hadjivassiliou et al (29) have proposed that IgG-AGA in sera from adult HLA-DQ2 and/or HLA-DQ8-positive patients with ataxia may cross-react with Purkinje cells in the cerebellum, leading to cerebellar ataxia, that is, gluten ataxia. In another study, they found that IgA-TG2 antibodies were present in the gut as well as in the brain of adult patients with gluten ataxia with or without signs of enteropathy (30). Recently, antibodies against neuronal TG6 were identified in patients with suspected gluten ataxia (31). The authors suggested that these antibodies may serve as an additional marker to identify a subgroup of patients with gluten sensitivity who may be at risk for developing neurological diseases with or without classical CD (31). In vitro studies have shown that TG6 can deamidate gluten peptides. It is still not known whether TG6 is expressed in the gut (32).
In conclusion, our findings support previous reports stating that routine small-bowel histology may not be sufficient to identify CD at an early stage, and that analysis of IgA colocalized with TG2 may prove useful when CD is suspected despite normal villous morphology. The majority of children with CP and elevated levels of CD-related seromarkers have neither classical nor early developing CD, but rather an increased immune reactivity to gluten. If there is any association between this so-called gluten reactivity, without signs of gut affection, then children's brain damage needs further studies.
1. Fung EB, Samson-Fang L, Stallings VA, et al. Feeding dysfunction is associated with poor growth and health status in children with cerebral palsy. J Am Diet Assoc
2. Day SM, Strauss DJ, Vachon PJ, et al. Growth patterns in a population of children and adolescents with cerebral palsy. Dev Med Child Neurol
3. Kuperminc MN, Stevenson RD. Growth and nutrition disorders in children with cerebral palsy. Dev Disabil Res Rev
4. Campanozzi A, Staiano A. Impact of malnutrition on gastrointestinal disorders and gross motor abilities in children with cerebral palsy. Brain Dev
5. Stenberg R, Dahle C, Lindberg E, et al. Increased prevalence of anti-gliadin antibodies and anti-tissue transglutaminase antibodies in children with cerebral palsy. J Pediatr Gastroenterol Nutr
6. Lahdeaho ML, Kaukinen K, Collin P, et al. Celiac disease: from inflammation to atrophy: a long-term follow-up study. J Pediatr Gastroenterol Nutr
7. Jarvinen TT, Kaukinen K, Laurila K, et al. Intraepithelial lymphocytes in celiac disease. Am J Gastroenterol
8. Salmi TT, Collin P, Jarvinen O, et al. Immunoglobulin A autoantibodies against transglutaminase 2 in the small intestinal mucosa predict forthcoming coeliac disease. Aliment Pharmacol Ther
9. Kurppa K, Collin P, Viljamaa M, et al. Diagnosing mild enteropathy celiac disease: a randomized, controlled clinical study. Gastroenterology
10. Korponay-Szabo IR, Halttunen T, Szalai Z, et al. In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies. Gut
11. Salmi TT, Collin P, Korponay-Szabo IR, et al. Endomysial antibody-negative coeliac disease: clinical characteristics and intestinal autoantibody deposits. Gut
12. Kaukinen K, Partanen J, Maki M, et al. HLA-DQ typing in the diagnosis of celiac disease. Am J Gastroenterol
13. Agardh D. Antibodies against synthetic deamidated gliadin peptides and tissue transglutaminase for the identification of childhood celiac disease. Clin Gastroenterol Hepatol
14. Sugai E, Vazquez H, Nachman F, et al. Accuracy of testing for antibodies to synthetic gliadin-related peptides in celiac disease. Clin Gastroenterol Hepatol
15. Karlberg J, Lawrence C, Albertsson-Wikland K. Prediction of final height in short, normal and tall children. Acta Paediatr Suppl 1994;406:3–9.
16. Palisano R, Rosenbaum P, Walter S, et al. Development and reliability of a system to classify gross motor function in children with cerebral palsy. Dev Med Child Neurol
17. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (“celiac sprue”). Gastroenterology
18. Oberhuber G, Granditsch G, Vogelsang H. The histopathology of coeliac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol Hepatol
19. Stoch MB, Smythe PM. The effect of undernutrition during infancy on subsequent brain growth and intellectual development. S Afr Med J
20. Hill PG, McMillan SA. Anti-tissue transglutaminase antibodies and their role in the investigation of coeliac disease. Ann Clin Biochem
21. Kaukinen K, Peraaho M, Collin P, et al. Small-bowel mucosal transglutaminase 2-specific IgA deposits in coeliac disease without villous atrophy: a prospective and randomized clinical study. Scand J Gastroenterol
22. Liden M, Kristjansson G, Valtysdottir S, et al. Gluten sensitivity in patients with primary Sjogren's syndrome. Scand J Gastroenterol
23. Lammers KM, Lu R, Brownley J, et al. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology
24. Clemente MG, De Virgiliis S, Kang JS, et al. Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function. Gut
25. Thomas KE, Sapone A, Fasano A, et al. Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in celiac disease. J Immunol
26. Tripathi A, Lammers KM, Goldblum S, et al. Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2. Proc Natl Acad Sci U S A
27. Villalta D, Alessio MG, Tampoia M, et al. Diagnostic accuracy of IgA anti-tissue transglutaminase antibody assays in celiac disease patients with selective IgA deficiency. Ann N Y Acad Sci
28. McGowan KE, Lyon ME, Butzner JD. Celiac disease and IgA deficiency: complications of serological testing approaches encountered in the clinic. Clin Chem
29. Hadjivassiliou M, Boscolo S, Davies-Jones GA, et al. The humoral response in the pathogenesis of gluten ataxia. Neurology
30. Hadjivassiliou M, Maki M, Sanders DS, et al. Autoantibody targeting of brain and intestinal transglutaminase in gluten ataxia. Neurology
31. Hadjivassiliou M, Aeschlimann P, Strigun A, et al. Autoantibodies in gluten ataxia recognize a novel neuronal transglutaminase. Ann Neurol
32. Stamnaes J, Dorum S, Fleckenstein B, et al. Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia. Amino Acids