A. Tosco

Clinical, HLA, and small bowel immunohistochemical features of children with positive serum antiendomysium antibodies and architecturally normal small intestinal mucosa.

BACKGROUND:Antiendomysium antibodies have a high sensitivity and specificity for celiac disease. A small percentage of subjects positive for these antibodies have a small intestinal mucosa hitherto considered normal.OBJECTIVES:The aim of this study was to characterize the clinical, serological, immunogenetic, and immunohistological features of these subjects.METHODS:From 409 patients who were positive for celiac-related antibodies, we selected 24 (5.9%) patients who had an architecturally normal small intestinal mucosa. One hundred age-matched celiac patients with a “flat” small intestinal mucosa, and 50 age-matched nonceliac children were also studied. The number of CD3+ and γδ+ intraepithelial lymphocytes and of CD25+ lamina propria mononuclear cells, and the expression of crypt HLA-DR and lamina propria ICAM-1 were assessed. HLA haplotyping was also performed.RESULTS:Eleven (45.8%) of the 24 patients had a distinct infiltrative pattern, i.e., an increase in CD3+ intraepithelial lymphocytes (> 2SD of the nonceliac group), whereas 17 (70.8%) had a higher density of intraepithelial γδ+ cells. In 17 (70.8%) patients, the number of lamina propria CD25+ cells was increased and/or the expression of ICAM-1 and crypt HLA-DR was enhanced. All 24 patients carried the celiac disease-associated HLA haplotypes. Two of the six patients who remained on a normal diet and underwent a second jejunal biopsy developed villous atrophy.CONCLUSIONS:Most of the patients with serum antiendomysium antibodies and normal jejunal histology showed immunohistochemical signs of immune activation in the epithelium, lamina propria, and crypts. We recommend that such patients be monitored to assess their progress and to determine whether they need a gluten-free diet.

Natural History of Potential Celiac Disease in Children

BACKGROUND & AIMS The presence of celiac disease-associated autoantibodies (antiendomysium and antitissue transglutaminase [anti-TG2]) with normal jejunal mucosa indicate potential celiac disease. We performed a prospective, 3-year cohort study to determine the natural history of potential celiac disease in children. METHODS The study included 106 children with potential celiac disease, based on serology analysis and normal duodenal architecture. All but 2 carried the HLA-DQ2 and/or DQ8 haplotype. In all children, every 6 months, growth, nutritional parameters, celiac disease serology, and autoimmunity were investigated. In biopsies, γδ intraepithelial-, CD3-, and lamina propria CD25-positive cells were counted; duodenal deposits of anti-TG2 immunoglobulin A were detected. Biopsy analysis was repeated after 2 years on patients with persistent positive serology and/or symptoms. RESULTS Celiac disease was detected primarily in first-degree relatives and patients with autoimmune disorders (40.6%). A gluten-free diet was prescribed to 20/106 patients because of symptoms, which were relieved in only 11. Eighty-nine of the 106 patients entered the follow-up study, with normal daily consumption of gluten. During the follow-up antibodies disappeared in 14.6% and fluctuated in 32.6%. Villous atrophy was observed in 12/39 patients (30.8%) who underwent a repeat biopsy. CONCLUSIONS Most children with potential celiac disease remain healthy. After 3 years, approximately 33% of patients develop villous atrophy. Intestinal deposits of anti-TG2 IgA identify children at risk for villous atrophy.

Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in CftrF508del homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the F508del-CFTR mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from F508del homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both in vivo, in mice, and in vitro, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 F508del-CFTR homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells in vivo, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of TNF/TNF-alpha (tumor necrosis factor) and CXCL8 (chemokine [C-X-C motif] ligand 8) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the F508del-CFTR mutation.

Immunoglobulin A anti-tissue transglutaminase antibody deposits in the small intestinal mucosa of children with no villous atrophy.

Objectives: Anti-tissue transglutaminase (anti-TG2) immunoglobulin A (IgA) autoantibodies are detectable in the serum of most patients with untreated celiac disease (CD). Their deposits in the intestine of patients with CD with severe enteropathy are considered specific for this condition. The histological spectrum of CD includes cases with normal villous architecture. The aim of this study was to look for anti-TG2 IgA deposits in the intestine of children with normal villous architecture and to relate them with other markers of gluten sensitivity. Patients and Methods: A total of 57 children with normal duodenal villous architecture and markers of gluten sensitivity were considered. Of those, 39 showed positive serum anti-endomysium antibodies and/or high levels of anti-TG2 antibodies (group 1), and 18 were seronegative with only a greater density of γδ intraepithelial lymphocytes (group 2). Thirty-four children with no markers of gluten sensitivity and a normal mucosa represented the control group (group 3). The duodenal sections of all patients were investigated for deposited anti-TG2 IgA by double immunofluorescence. Human lymphocyte antigen molecular typing was performed. Results: In group 1 and in group 2, 33 of 39 children (85%) and 12 of 18 children (66%) showed subepithelial anti-TG2 IgA intestinal deposits, respectively. Only in 3 of 34 (8.8%) children with no markers of gluten sensitivity were anti-TG2 IgA deposits noted. Conclusions: A subgroup of children with no serum CD-associated autoantibodies, but greater density of γδ intraepithelial lymphocytes, shows a clear anti-TG2 IgA deposition in the duodenal mucosa. These children must be investigated further for possible gluten sensitivity.

Potential Celiac Children: 9-Year Follow-Up on a Gluten-Containing Diet

OBJECTIVES:Potential celiac disease (CD) is defined by the presence of serum anti-tissue-transglutaminase (anti-TG2) antibodies and normal duodenal mucosa. The major clinical problem is the management of asymptomatic patients and how to predict the development of villous atrophy. This prospective longitudinal cohort study describes the natural history of potential CD up to 9 years and explores risk factors associated with the development of mucosal damage.METHODS:Two hundred and ten potential CD children were eligible for the study; 175/210 asymptomatic children were left on a gluten-containing diet. Antibodies and clinical symptoms were checked every 6 months, and a small bowel biopsy was taken every 2 years to evaluate histological, immunohistochemical, and anti-TG2 deposits. Patients were genotyped for HLA and a set of non-HLA CD-associated genes.RESULTS:Forty-three percent of patients showed persistently elevated anti-TG2 level, 20% became negative during follow-up, and 37% showed a fluctuant anti-TG2 course with transiently negative values. At 3 years of follow-up, 86% of cases remained potential; 73 and 67% still had normal duodenal architecture at 6 and 9 years, respectively. Male sex, slight mucosal inflammation at time 0, and a peculiar genetic profile delineate a cohort of individuals who were prone to develop mucosal damage during time.CONCLUSIONS:A sizeable proportion of asymptomatic potential celiac patients showed fluctuation or negativization of antibody production, and many of these, with persistently positive anti-TG2, did not develop mucosal damage after 9 years of follow-up. Celiac population is a multivariate aggregate of individuals with different genetic and phenotypic profiles. Caution is required before prescribing a gluten-free diet for life to asymptomatic individuals with potential CD.

Potential Celiac Patients: A Model of Celiac Disease Pathogenesis

Background and Aim Potential celiacs have the ‘celiac type’ HLA, positive anti-transglutaminase antibodies but no damage at small intestinal mucosa. Only a minority of them develops mucosal lesion. More than 40 genes were associated to Celiac Disease (CD) but we still do not know how those pathways transform a genetically predisposed individual into an affected person. The aim of the study is to explore the genetic features of Potential CD individuals. Methods 127 ‘potential’ CD patients entered the study because of positive anti-tissue transglutaminase and no mucosal lesions; about 30% of those followed for four years become frankly celiac. They were genotyped for 13 polymorphisms of ‘candidate genes’ and compared to controls and celiacs. Moreover, 60 biopsy specimens were used for expression studies. Results Potential CD bear a lighter HLA-related risk, compared to celiac (χ2 = 48.42; p value = 1×10−8). They share most of the polymorphisms of the celiacs, but the frequency of c-REL* G allele was suggestive for a difference compared to celiac (χ2 = 5.42; p value = 0.02). One marker of the KIAA1109/IL-2/IL-21 candidate region differentiated potentials from celiac (rs4374642: χ2 = 7.17, p value = 0.01). The expression of IL-21 was completely suppressed in potentials compared to celiacs (p value = 0.02) and to controls (p value = 0.02), in contrast IL-2, KIAA1109 and c-REL expression were over-expressed. Conclusions Potential CD show genetic features slightly different from celiacs. Genetic and expression markers help to differentiate this condition. Potential CD is a precious biological model of the pathways leading to the small intestinal mucosal damage in genetically predisposed individuals.

Serum and intestinal celiac disease-associated antibodies in children with celiac disease younger than 2 years of age.

Objectives: In children younger than 2 years of age, a diagnosis of celiac disease (CD) is difficult to make because anti-endomysium (anti-EMA)/anti-tissue transglutaminase 2 (anti-TG2) antibodies are less sensitive than in older children. The aim of our study was to evaluate how many children younger than 2 years of age and diagnosed with CD, were negative for serum anti-TG2 antibodies and to test the hypothesis that in these patients, TG2-specific IgA deposits could instead be present at mucosal level. Patients and Methods: A total of 104 children younger than 2 years of age and 179 children older than 2 years, all of whom had been diagnosed with CD, were investigated for serum CD-associated antibodies (anti-gliadin [AGA] IgA and IgG, EMA-IgA, anti-TG2–IgA). The presence of intestinal anti-TG2 extracellular IgA deposits was searched by using double immunofluorescence in 56 of the patients younger than 2 years of age and in 40 of those who were older than 2 years. Results: In children with CD who were younger than 2 years of age, high levels of AGA-IgA were found in 93/104 (89%) and 98/104 (94%) were found of have high levels of AGA-IgG. In children older than the age of 2 years with CD, 120/179 (67%) had high levels of AGA-IgA and 151/179 (84%) had high levels of AGA-IgG. Serum EMA were present in 92/104 (88%) in the younger group and in 176/179 (98%) of the older group. Ninety-one of 104 children (87%) younger and 172/179 (96%) older than 2 years showed high serum levels of anti-TG2. Finally, 41/56 (73%) children younger than 2 years and all of the 40 children (100%) older than 2 years of age showed mucosal anti-TG2–IgA deposits. Conclusions: EMA and anti-TG2–antibody measurements show higher sensitivity for the diagnosis of CD in children older than 2 years compared with younger children. The search for mucosal deposits of anti-TG2–IgA does not improve the diagnostic performance.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts ‘on-target’ because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Intestinal deposits of anti-tissue transglutaminase IgA in childhood celiac disease

BACKGROUND High serum levels of anti-tissue-transglutaminase-2 IgA antibodies (anti-TG2), which are produced and deposited in the intestine, characterize celiac disease. AIM To assess the diagnostic value of intestinal deposits of anti-TG2 IgA for celiac disease in a paediatric population. METHODS 344 children underwent duodenal biopsy for the suspicion of CD, and were divided into 3 groups: group A, 144 celiac subjects with villous atrophy (Marsh 3b-c); group B, 109 subjects with high serum levels of anti-TG2 but normal intestinal mucosa (Marsh 0-1) (potential celiac disease patients); group C, 91 subjects with normal levels of serum anti-TG2: 70 with Marsh 0-1 and 21 with Marsh 3a mucosa. All duodenal sections were evaluated for the presence of intestinal deposits of anti-TG2 IgA by double immunofluorescence. RESULTS Deposits of anti-TG2 IgA were present in 96%, 68%, 12% of patients from groups A, B, C, respectively. Diagnostic sensitivity and specificity for celiac disease were 96% and 88% vs. 97% and 100% for serum anti-TG2, respectively. The degree of concordance with serum anti-TG2 was 85%. CONCLUSION Detection of intestinal deposits of anti-TG2 IgA is a useful diagnostic tool. Further research is needed regarding their ability to predict evolution to villous atrophy in potential celiac disease.

Cysteamine re-establishes the clearance of Pseudomonas aeruginosa by macrophages bearing the cystic fibrosis-relevant F508del-CFTR mutation

Cystic fibrosis (CF), the most common lethal monogenic disease in Caucasians, is characterized by recurrent bacterial infections and colonization, mainly by Pseudomonas aeruginosa, resulting in unresolved airway inflammation. CF is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein, which functions as a chloride channel in epithelial cells, macrophages, and other cell types. Impaired bacterial handling by macrophages is a feature of CF airways, although it is still debated how defective CFTR impairs bacterial killing. Recent evidence indicates that a defective autophagy in CF macrophages leads to alterations of bacterial clearance upon infection. Here we use bone marrow-derived macrophages from transgenic mice to provide the genetic proof that defective CFTR compromises both uptake and clearance of internalized Pseudomonas aeruginosa. We demonstrate that the proteostasis regulator cysteamine, which rescues the function of the most common F508del-CFTR mutant and hence reduces lung inflammation in CF patients, can also repair the defects of CF macrophages, thus restoring both bacterial internalization and clearance through a process that involves upregulation of the pro-autophagic protein Beclin 1 and re-establishment of the autophagic pathway. Altogether these results indicate that cysteamine restores the function of several distinct cell types, including that of macrophages, which might contribute to its beneficial effects on CF.