Redefining the intraepithelial lymphocytes threshold to diagnose gluten sensitivity in patients with architecturally normal duodenal histology.

BACKGROUND: Accuracy of intraepithelial lymphocytes counts for diagnosing mild enteropathy coeliac disease in absence of villous atrophy can be limited by inappropriate controls included in the studies. AIM: To determine the diagnostic accuracy of intraepithelial lymphocytes counts utilising controls lacking HLA coeliac disease-associated alleles. METHODS: Intraepithelial lymphocytes counting at villus tip and per 100 enterocytes was performed at haematoxylin and eosin (H&E) and CD3-stainings in: 29 cases (21 with potential coeliac disease and 8 affected by latent coeliac disease) representing the patient population and 14 noncoeliac controls lacking HLA-DQ2/DQ8 alleles. RESULTS: Threshold (mean+2 s.d.) of duodenal intraepithelial lymphocytes at villus tip and per 100 enterocytes in noncoeliac controls was respectively: 3.5 and 18 at H&E, 3.2 and 17 following CD3-staining. Considering the whole patient population, the sensitivity of tip intraepithelial lymphocytes in detecting mild enteropathy coeliac disease was 90% (95% CI=72.6-97.8) both at H&E and CD3-stainings. The sensitivity of intraepithelial lymphocytes per 100 enterocytes was 93% (95% CI=77.2-99.2) both at H&E and CD3-staining. Specificity of both intraepithelial lymphocytes counts was 100% (95% CI=76.8-100). Using a threshold of 25 intraepithelial lymphocytes per 100 enterocytes could miss 59% of cases at H&E and 48% following CD3-staining. CONCLUSIONS: Intraepithelial lymphocytes counts are diagnostic feasible tools to detect mild enteropathy coeliac disease. Threshold of duodenal intraepithelial lymphocytes may be lower than currently accepted.

Comparison of the Marsh-Oberhuber classification with a new grading system in identifying patients with latent celiac disease.

AIM: The diagnosis of celiac disease (CD) is still mainly based on pathological description. However, these descriptions are often unable to identify latent CD. The aim of this study was to evaluate whether the Marsh-Oberhuber classification and a recently proposed classification may help to identify patients with latent CD. METHODS: Biopsy samples from twelve patients with latent CD (age range 3-32 years) defined as having normal duodenal mucosa when ingesting a free diet, and subsequently developing severe villous atrophy, were retrospectively reviewed in blind according to the Marsh-Oberhuber classification and the new grading system. RESULTS: In 67% of patients the Marsh-Oberhuber and the new classification could have yielded a diagnosis of CD soon after the first biopsy (3a-3c score when reviewed according to this classification, and B2 score when reviewed according to the new grading system), thereby avoiding further (up to two more in four cases) unnecessary endoscopic procedures. CONCLUSION: Both the Marsh-Oberhuber and the new classification allow to discriminate latent CD from patients with normal mucosa. Thus, these classifications may help in identifying and treating patients at an early stage.

Inflammatory bowel disease in children and adolescents in Italy: data from the pediatric national IBD register (1996-2003).

BACKGROUND: The purpose was to assess in Italy the clinical features at diagnosis of inflammatory bowel disease (IBD) in children. METHODS: In 1996 an IBD register of disease onset was established on a national scale. RESULTS: Up to the end of 2003, 1576 cases of pediatric IBD were recorded: 810 (52%) ulcerative colitis (UC), 635 (40%) Crohn's disease (CD), and 131 (8%) indeterminate colitis (IC). In the period 1996-2003 an increase of IBD incidence from 0.89 to 1.39/10(5) inhabitants aged <18 years was observed. IBD was more frequent among children aged between 6 and 12 years (57%) but 20% of patients had onset of the disease under 6 years of age; 28 patients were <1 year of age. Overall, 11% had 1 or more family members with IBD. The mean interval between onset of symptoms and diagnosis was higher in CD (10.1 months) and IC (9 months) versus UC (5.8 months). Extended colitis was the most frequent form in UC and ileocolic involvement the most frequent in CD. Upper intestinal tract involvement was present in 11% of CD patients. IC locations were similar to those of UC. Bloody diarrhea and abdominal pain were the most frequent symptoms in UC and IC, and abdominal pain and diarrhea in CD. Extraintestinal symptoms were more frequent in CD than in UC. CONCLUSIONS: The IBD incidence in children and adolescents in Italy shows an increasing trend for all 3 pathologies. UC diagnoses exceeded CD.

Menarcheal age in celiac disease may not be delayed and may be irrespective of age at diagnosis and dietary management.

The aim of the present study was to evaluate the role played by age at diagnosis of celiac disease (CD), dietary management and menarcheal familiar antecedents in conditioning menarcheal age (MA) in CD. This study covers a population of 94 menarcheal adolescents with untreated CD, whose MA was compared with that of 3 control populations: the 1st consisting of 117 early-treated and compliant CD girls, the 2nd represented by their non-celiac mothers, and the 3rd consisting of 280 healthy adolescents. Average MA of the girls with post-menarcheal diagnosis of CD was superimposable to that of the patients with pre-menarcheal diagnosis and was no different from the one of their mothers or that of healthy controls. The prevalence of delayed menarche was similar in the patients with either pre-menarcheal or post-menarcheal diagnosis of CD. A direct correlation between patients' MA and that of their mothers was detected in both groups of CD patients. We conclude that: a) untreated CD may not be associated with menarcheal retardation; b) MA in CD is significantly affected by maternal MA and may be irrespective of age at diagnosis and dietary management.

Concordance, disease progression, and heritability of coeliac disease in Italian twins.

BACKGROUND AND AIMS: We adopted the twin method to disentangle the genetic and environmental components of susceptibility to coeliac disease (CD). We estimated disease concordance rate by zygosity and HLA genotypes, discordance times, progression rates to disease, and heritability. METHODS: We crosslinked the Italian Twin Registry with the membership lists of the Italian Coeliac Disease Association and recruited 23 monozygotic (MZ) and 50 dizygotic (DZ) twin pairs with at least one affected member. Zygosity was assigned by DNA fingerprinting, and HLA-DQ and DR alleles were genotyped. Disease status was ascertained by antiendomysial, anti-human tissue transglutaminase antibodies, and bowel biopsy. RESULTS: Concordance was significantly higher in MZ (83.3% probandwise, 71.4% pairwise) than in DZ (16.7% probandwise, 9.1% pairwise) pairs. Concordance was not affected by sex or HLA genotype of the co-twin and being MZ was significantly associated with the occurrence of CD (Cox adjusted hazard ratio 14.3 (95% confidence interval 4.0-50.3)). In 90% of concordant pairs the discordance time was

Chronic urticaria and associated coeliac disease in children: a case-control study.

UNLABELLED: Celiac disease (CD) and chronic urticaria (CU) are both sustained by immune mechanisms, but there are so far few data on their clinical association. We performed a case-control study to determine the occurrence of CD in urticaria and matched control children, and to assess the clinical relevance of this association. Children and adolescents were diagnosed to have severe chronic idiopathic urticaria in the presence of hives for more than 6 wk poorly or not responsive to oral antihistamines. Other known causes of urticaria had to be excluded. A matched control group without urticaria was enrolled. In both groups, the presence of CD was searched by assaying antitransglutaminase and antiedomysial antibodies, and confirmed with endoscopic intestinal biopsy. Results. CD was diagnosed and confirmed in 4/79 (5.0%) of children with CU and in 17/2545 (0.67%) of the controls (p = 0.0003). In the four children with urticaria and CD the gluten free diet (GFD) lead to complete remission of urticaria within 5-10 wk, whereas the disappearance of serological markers occurred in longer times (5-9 months). CONCLUSIONS: The presence of CD in children with CU was significantly more frequent than in controls. GFD resulted in urticaria remission. CD may be regarded in such subjects as a cause of CU.

Usefulness of omega-3 fatty acid supplementation in addition to mesalazine in maintaining remission in pediatric Crohn's disease: a double-blind, randomized, placebo-controlled study.

AIM: To assess the value of long-chain omega-3 fatty acids (FAs) supplementation in addition to amino-salicylic-acid (5-ASA) in pediatric patients with Crohn's disease (CD). METHODS: Thirty-eight patients (20 males and 18 females, mean age 10.13 years, range 5-16 years) with CD in remission were randomized into two groups and treated for 12 mo. Group I (18 patients) received 5-ASA (50 mg/kg/d)+ omega-3 FAs as triglycerides in gastro-resistant capsules, 3 g/d (eicosapentanoic acid, EPA, 400 mg/g, docosahexaenoic acid, DHA, 200 mg/g). Group II (20 patients) received 5-ASA (50 mg/kg/d)+olive oil placebo capsules. Patients were evaluated for fatty acid incorporation in red blood cell membranes by gas chromatography at baseline 6 and 12 mo after the treatment. RESULTS: The number of patients who relapsed at 1 year was significantly lower in group I than in group II (P<0.001). Patients in group I had a significant increase in the incorporation of EPA and DHA (P<0.001) and a decrease in the presence of arachidonic acids. CONCLUSION: Enteric-coated omega-3 FAs in addition to treatment with 5-ASA are effective in maintaining remission of pediatric CD.

Increased frequency of the immunoglobulin enhancer HS1,2 allele 2 in coeliac disease.

BACKGROUND: Coeliac disease (CD) is characterized by increased immunological responsiveness to ingested gliadin in genetically predisposed individuals. This genetic predisposition is not completely defined. A dysregulation of immunoglobulins (Ig) is present in CD: since antiendomysium antibodies (anti-EMA) are of the IgA class. One polymorphic enhancer within the locus control region (LCR) of the immunoglobulin heavy chain cluster at the 3' of the C alpha-1 gene was investigated. The correlation of the penetrance of the four different alleles of the HS1,2-A enhancer of the LCR-1 3' to C alpha-1 in CD patients compared to a control population was analysed. METHODS: A total of 115 consecutive CD outpatients, on a gluten-free diet, and 248 healthy donors, age- and sex-matched, from the same geographical area were enrolled in the study. HS1,2-A allele frequencies were investigated by nested polymerase chain reaction (PCR). RESULTS: The frequency of allele 2 of the enhancer HS1,2-A gene was increased by 30.8% as compared to the control frequency. The frequency of homozygosity for allele 2 was significantly increased in CD patients. Crude odds ratio (OR) showed that those with 2/2 and 2/4 (OR 2.63, P < 0.001 and OR 2.01, P = 0.03) have a significantly higher risk of developing the disease. In contrast, allele 1/2 may represent a protective genetic factor against CD (OR 0.52, P = 0.01). CONCLUSIONS: These data provide further evidence of a genetic predisposition in CD. Because of the Ig dysregulation in CD, the enhancer HS1,2-A may be involved in the pathogenesis.

Natural history of glucose tolerance, beta-cell function and peripheral insulin sensitivity in cystic fibrosis patients with fasting euglycemia.

OBJECTIVE: The loss of pancreatic beta-cells is thought to be one of the principal causes of diabetes mellitus (DM) in cystic fibrosis (CF), but the role of peripheral insulin resistance (IR) in the pathogenesis of DM in CF remains unclear. The aim of this study was to evaluate whether eventual changes of glucose tolerance (GT) over time were associated with modifications of insulin secretion or sensitivity. METHODS: Plasma glucose and insulin responses to an oral GT test (OGTT) were investigated and reinvestigated 13 Years later in 14 CF patients with initial and persistent fasting euglycemia and no history of insulin treatment. Insulin sensitivity (IS) at both tests was assessed on the basis of insulin and glucose levels both in the fasting state and during OGTTs. RESULTS: From the 1st to the 2nd OGTT: (a) the prevalence of DM responses significantly increased; (b) the areas beneath the respective glucose and insulin curves significantly increased and decreased respectively; (c) IR and IS indices decreased and increased respectively, even in the patients who developed DM; (d) pulmonary function significantly worsened in the entire series, especially in the patients who developed DM. CONCLUSIONS: (i) the natural history of glyco-metabolic status in CF is characterized by deteriorating GT over time; (ii) insulinopenia plays a prominent role in the pathogenesis of GT worsening; (iii) IR does not play any significant part in the pathogenesis of DM development; (iv) deterioration of lung function tests is more severe in the subjects who develop DM over time.

Pubertal development in cystic fibrosis: an overview.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by respiratory and intestinal insufficiencies. It has been reported in the literature that patients with CF show delayed growth and puberty and girls with CF achieve menarche at older age than normal females. Infertility, in both sexes, and menstrual dysfunction, in girls, are common in patients with CF. Previous data suggest that the degree of failure of growth and development is correlated with malnutrition and severity of progressing pulmonary disease. Despite improved nutrition and intensive treatment, patients with CF have delayed puberty and growth pubertal spurt. This maturational lag is accompanied by a significant delay in attaining pubertal levels of insulin-like growth factor-I (IGF-I), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and sex steroid hormones.

Factors affecting diabetes mellitus onset in cystic fibrosis: evidence from a 10-year follow-up study.

This study reports the results of genotype characterization and of a 10-y prospective evaluation of clinical status, glucose tolerance and insulin secretion in 28 originally normoglycaemic patients with cystic fibrosis (CF). The aim of the study was to assess whether any genetic, clinical or metabolic parameters could identify in advance those patients at risk of developing diabetes mellitus over time. During the follow-up 42.8% of patients became diabetic. Neither gender, age nor clinical parameters were significantly different at entry in the patients who eventually developed diabetes compared with those who did not. Insulin secretion during oral glucose tolerance tests (OGTT) deteriorated over time in both groups, whereas a progressive deterioration of glucose tolerance was only evident in the patients who developed diabetes and increased baseline glucose areas were the only predictive parameter of diabetes onset. Genotype analysis revealed significant differences between patients with and without diabetes: deltaF508 homozygosis was more frequent in the first group and N1303K mutation in the second group. In conclusion, in CF: (i) increased glucose areas during OGTT and deterioration of glucose tolerance over time can predict the evolution towards diabetes; and (ii) deltaF508 homozygosis may predispose to the risk of diabetes, whilst N1303K mutation seems to play a protective role.

Bone demineralization in cystic fibrosis: evidence of imbalance between bone formation and degradation.

Bone turnover, collagen metabolism, and bone mineral status were investigated in 59 patients with cystic fibrosis and in 72 sex and age-matched control subjects. In all patients and control subjects serum concentrations of osteocalcin (OC), carboxy-terminal propeptide of type I procollagen (PICP), amino-terminal propeptide of type III procollagen (PIIINP), and cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), and urinary values of cross-linked N-telopeptides of type I collagen (NTX), as well as total body bone mineral content (TBBM) were measured. Higher ICTP (microgram/L) and NTX (bone collagen equivalent/urinary creatinine (nmol/mmol) values were found in pre-pubertal, pubertal, and young adult patients than in control subjects (ICTP: 15.4 +/- 2.1 and 13.2 +/- 1.8, p < 0.001; 23.3 +/- 5.3 and 20.1 +/- 4.1, p < 0.02; 4.8 +/- 1.1 and 4.0 +/- 1.0, p < 0.05. respectively; NTX: 1047.5 +/- 528.6 and 227.8 +/- 71.8, p < 0.01; 997.8 +/- 391.7 and 376.3 +/- 91.0, p < 0.01; 993.2 +/- 398.0 and 73.9 +/- 28.5, p < 0.01, respectively). Lower OC and PICP levels (microgram/L) were showed in pubertal patients in comparison with control subjects (OC: 20.2 +/- 12.3 and 39.0 +/- 15.1, p < 0.01; PICP: 305.8 +/- 130.4 and 436.2 +/- 110.1, p < 0.02, respectively). Lower OC and higher PIIINP levels (microgram/L) were found in young adult patients than in control subjects (OC: 4.4 +/- 3.0 and 7.0 +/- 3.1, p < 0.05; PIIINP: 4.8 +/- 1.1 and 3.1 +/- 1.0, p < 0.001, respectively). TBBM (z score) was reduced in prepubertal, pubertal, and young adult patients (-0.8 +/- 0.4, -1.0 +/- 0.4, -1.1 +/- 0.5, respectively). Patients with cystic fibrosis have bone demineralization and imbalance between bone formation and degradation.

Metabolic and clinical events preceding diabetes mellitus onset in cystic fibrosis.

Serial assessments of glucose tolerance, of glucose and insulin areas during an oral glucose tolerance test (OGTT) and of clinical parameters as well were evaluated retrospectively in seven diabetic cystic fibrosis patients (study group) during the 4-6.3 years that preceded diabetes mellitus diagnosis. The same metabolic and clinical parameters were also evaluated in seven age-matched patients who did not develop diabetes during a similar observation period (control group). In the study group, glucose tolerance was impaired in all patients but one since the first OGTT and glucose areas progressively increased over time, whereas in the control group glucose tolerance remained stable during the whole observation period. A significant and progressive blunting of insulin secretion occurred over time in both groups. Insulin secretion, however, was reduced but not exhausted at diabetes diagnosis. Neither modification of glycosylated haemoglobin levels over time nor serum islet cell antibodies at diabetes onset were observed in the study group. The overall clinical course of the disease was not different in either group and remained stable during the observation period. These results indicate that in cystic fibrosis diabetes mellitus onset is preceded by a long-standing deterioration of glucose tolerance, whilst insulin secretion progressively declines over time, irrespectively of glucose tolerance status. The prediabetic worsening of glucose tolerance is not necessarily linked to a worsening of overall clinical status.

Four-year follow-up of glucose tolerance and beta-cell function in nondiabetic cystic fibrosis patients.

Plasma glucose and insulin responses to oral glucose load were investigated and reinvestigated approximately 4 years later in 29 cystic fibrosis children and adolescents with initially normal fasting blood glucose levels. Patients' clinical status was evaluated at the time of both oral glucose tolerance tests. With respect to the basal test, the second one elicited blunted insulin responses and enhanced glycemic levels. Moreover, the prevalence of patients with diabetic glucose tolerance was significantly increased at the second evaluation and insulin secretion was markedly reduced in these patients. Deterioration of glucose tolerance and/or of insulin secretion over time was never accompanied by a significant worsening of clinical and/or nutritional status. In conclusion, in cystic fibrosis subjects with fasting euglycemia (a) both insulin secretion and glucose tolerance deteriorate during a 4-year follow-up, (b) an insulin secretion decrease is more evident in patients who develop diabetic glucose tolerance and (c) these metabolic changes are not significantly linked to a worsening of either nutritional or clinical parameters, even in the patients who develop diabetic glucose tolerance.

No changes of insulin sensitivity in cystic fibrosis patients with different degrees of glucose tolerance: an epidemiological and longitudinal study.

Plasma glucose and insulin responses to oral glucose and insulin sensitivity by the euglycemic hyperinsulinemic clamp technique were investigated in 30 cystic fibrosis patients with normal fasting blood glucose levels and normal (N = 12), impaired (N = 12) or diabetic (N = 6) glucose tolerance, and in 12 control subjects. In a subgroup of 10 cystic fibrosis patients with non-diabetic glucose tolerance both oral glucose tolerance test and clamp were performed again 48-52 months later. Following oral glucose, glycemic responses were higher in cystic fibrosis patients than in controls, whereas insulin responses were reduced significantly only in the patients with diabetic glucose tolerance. Insulin sensitivity did not differ significantly in the patient subgroups with different degrees of glucose tolerance and in controls. In the 10 patients who underwent a 4-year follow-up, insulin responses to oral glucose decreased significantly, whilst insulin sensitivity did not change substantially. Insulin sensitivity persisted unmodified even in the patients with deteriorating glucose tolerance. No correlation were observed between metabolic data and clinical status of patients. In conclusion, in cystic fibrosis subjects with fasting euglycemia and different degrees of glucose tolerance: (i) insulin sensitivity is not impaired; (ii) eventually changes of glucose tolerance over time are not associated with modifications of insulin sensitivity; (iii) insulin secretion deteriorates over time even in the patients with stable glucose tolerance; (iv) eventual deterioration of both glucose tolerance and insulin secretion is not linked to a worsening of either nutritional or clinical parameters.

First-phase insulin response to intravenous glucose in cystic fibrosis patients with different degrees of glucose tolerance.

The aim of our study was to determine whether first-phase insulin response to intravenous (i.v.) glucose could be used as a simple and rapid test to identify cystic fibrosis (CF) patients at risk to develop diabetes mellitus. Forty consecutive CF patients with normal fasting blood glucose values but with different degrees of glucose tolerance on the standard oral glucose tolerance test (22 with normal glucose tolerance, 16 with impaired glucose tolerance, 2 with diabetes mellitus) and 12 normal subjects, matched for age and body mass index, underwent an i.v. glucose bolus to evaluate early phase insulin release. When compared to the normal subjects, CF patients had significantly reduced basal (76 +/- 50 vs 108 +/- 30 pM/l, 2 p < 0.02) and glucose stimulated insulin levels (1 + 3 min insulin = 456 +/- 275 vs 951 +/- 170 pM/l, 2 p < 0.01). Early phase insulin release, however, did not differentiate between CF patients with normal and impaired glucose tolerance; also in the two diabetic patients insulin levels did not clearly differ from those observed in the other groups of CF subjects. In conclusion, first-phase insulin response may identify an impairment of B-cell function in CF subjects; however, it does not discriminate between different degrees of glucose tolerance, as determined by the oral glucose tolerance test and, therefore, it does not reliably identify those patients who will eventually develop overt diabetes mellitus.

Insulin secretion, glycosylated haemoglobin and islet cell antibodies in cystic fibrosis children and adolescents with different degrees of glucose tolerance.

In comparison with 12 weight-matched controls, 39 children and adolescents with cystic fibrosis (CF) showed higher fasting glycaemic levels and both delayed and enhanced blood glucose responses to OGTT. Glycaemic response was normal in 30/39 patients (76.9%), impaired in other 7 cases (18%) and diabetic in the remnant two (5.1%). Fasting insulin levels and total insulin output during OGTT did not differ in patients and controls, but insulin peak in CF group was delayed and sustained. In the whole CF series mean HbA1c was higher than in controls but no difference was found between patients with normal and those with impaired glucose tolerance. Islet cell antibodies were absent in the entire CF group. In conclusion, our results confirm the raised prevalence in CF of glucose tolerance abnormalities, which do not seem to depend on auto-immune factor involvement. Delayed insulin response to OGTT can be considered a very early expression of beta cell impairment in the course of CF. In our experience HbA1c assay did not constitute a sensitive and specific screening test for detection of the C patients with glucose intolerance.