Non-organ-specific autoimmunity in adult 47,XXY Klinefelter patients and higher-grade X-chromosome aneuploidies.

Current literature regarding systemic autoimmune diseases in X-chromosome aneuploidies is scarce and limited to case reports. Our aim was to evaluate the frequency of anti-nuclear (ANAs), extractable nuclear (ENA), anti-double-stranded DNA (dsDNAs), anti-smooth muscle (ASMAs) and anti-mitochondrial (AMAs) antibodies in a large cohort of adults with Klinefelter's syndrome (KS, 47,XXY) and rare higher-grade sex chromosome aneuploidies (HGAs) for the first time. Sera from 138 X-chromosome aneuploid patients [124 adult patients with 47,XXY KS and 14 patients with HGA (six children, eight adults)] and 50 age-matched 46,XY controls were recruited from the Sapienza University of Rome (2007-17) and tested for ANAs, ENAs, anti-dsDNAs, ASMAs and AMAs. Non-organ-specific immunoreactivity was found to be significantly higher in patients with 47,XXY KS (14%) than in the controls (2%, p = 0.002). Among all the antibodies investigated, only ANAs were observed significantly more frequently in patients with 47,XXY KS (12.1%) than in the controls (2%, p = 0.004). No anti-dsDNA immunoreactivity was found. Stratifying by testosterone replacement therapy (TRT), non-organ-specific autoantibody frequencies were higher in TRT-naive (p = 0.01) and TRT-treated groups than in controls. No patients with HGA were found positive for the various autoantibodies. Non-organ-specific autoantibodies were significantly present in 47,XXY adult patients. Conversely, HGAs did not appear to be target of non-organ-specific immunoreactivity, suggesting that KS and HGAs should be considered as two distinct conditions. The classification and diagnosis of systemic autoimmune diseases is frequently difficult. To support a correct clinical evaluation of KS disease and to prevent eventual secondary irreversible immune-mediated damages, we highlight the importance of screening for non-organ-specific autoimmunity in Klinefelter's syndrome.

Gluten-free diet impact on dynamics of pancreatic islet-specific autoimmunity detected at celiac disease diagnosis.

OBJECTIVE: Almost 6% of celiac disease (CD) patients at diagnosis are positive for at least one of the main pancreatic islet autoantibodies that characterize type 1 diabetes (T1D). Few information, dated back to almost two decades ago, exist as to whether a gluten-free diet (GFD) could reduce the islet-specific autoimmunity detected in patients at CD diagnosis. Aim of the study was to evaluate the impact of GFD on 31 patients who presented islet-specific autoimmunity at CD diagnosis. METHODS: CD patient sera collected at diagnosis and throughout the GFD were analyzed for the main humoral autoantibodies so far identified in T1D, directed against one or more among insulin, glutamic-acid decarboxylase, tyrosine-phosphatase 2, and zinc cation-efflux transporter autoantigens. RESULTS: GFD (median duration 39 months) was associated to a decrease or disappearance of the islet-specific autoantibodies in 71% of CD patients. Almost 80% of the patients who became autoantibody-negative during the GFD were positive for only one of the islet-specific autoimmune markers at CD diagnosis, with none of them developing diabetes. Conversely, 80% of the CD patients positive at diagnosis for ≥2 islet-specific autoantibodies were still positive after more than two years of GFD, with 25% of them developing T1D. CONCLUSIONS: Various factors appear to influence, individually or in combination, the effects of the GFD on pancreatic islet-specific autoimmune response detected at CD diagnosis. These factors include the number of diabetes autoantibodies found at CD diagnosis, the adherence to the GFD, its duration and an asymptomatic clinical presentation of CD.

Type 1 diabetes, thyroid, gastric and adrenal humoral autoantibodies are present altogether in almost one third of adult celiac patients at diagnosis, with a higher frequency than children and adolescent celiac patients.

Background: No data are available on the frequency of organ-specific humoral autoimmunity at diagnosis of adult celiac disease (CD).Aim: To evaluate the humoral immunoreactivities specific of type 1 diabetes (T1D), thyroid (THD), atrophic-gastritis (AG) and Addison's (AD) diseases in 92 adult CD patients at diagnosis and 237 adult healthy subjects (CTRL).Methods: T1D, THD and AD specific autoantibodies were analyzed by radioimmunoprecipitation assays. AG autoantibodies were detected by enzyme-linked immunosorbent assay.Results: Of 92 CD patients, 31.5% were positive for at least one of the organ-specific autoantibodies investigated (p < .0001 vs CTRL). Thyroid, diabetes, gastric and adrenal-autoantibodies, that increase with age at diagnosis, were detected in 12.0%, 10.9%, 10.9%, 2.2% of CD patients, respectively. Gastric- and diabetes- rather than thyroid- and adrenal-autoimmunity seem to be specifically related to presence of CD.Conclusions: One third of adult CD patients at diagnosis is target of at least one organ-specific autoantibody. A systematic organ-specific autoantibody screening in these patients might be of value to promptly identify, prevent or treat the relative diseases.

Evidence of diabetes-specific autoimmunity in obese subjects with normal glucose tolerance.

BACKGROUND: Recently, significant attention has been paid to the possible activation of an autoimmune response in the presence of obesity. The aim of this study was to evaluate and compare the frequencies of autoantibodies typical of autoimmune diabetes in obese patients with normal glucose tolerance (NGT), obese patients with type 2 diabetes (T2D) and controls. We also evaluated the presence of immunoreactivity to Hashimoto's thyroiditis and autoimmune gastritis. MATERIALS AND METHODS: Consecutive sera from obese patients, 444 with NGT, 322 with T2D, and 212 controls were analysed by radioimmunoassay or enzyme-linked immunosorbent assay for glutamic acid decarboxylase, protein tyrosine phosphatase islet antigen-2 (IA-2)IC and IA-2(256-760) , islet beta-cell zinc cation transporter (ZnT8), thyroid peroxidase, and anti-parietal cell autoantibodies. RESULTS: Altogether the presence of organ-specific autoantibodies was significantly more frequent in obese patients with NGT (128/444, 28.5%) and obese with T2D (79/322, 24.5%) than in controls (36/212, 17%; P = 0.002). Thyroid peroxidase immunoreactivity was prevalent in all groups of subjects investigated. The frequencies of diabetes-specific autoantibodies were slightly higher in obese patients with NGT (20/444, 4.5%) than in obese with T2D (12/322, 3.7%) and controls (4/212, 1.9%). The anti IA-2(256-760) was the most frequent islet autoantibody in obese subjects with NGT (14/20, 70%). CONCLUSIONS: We observed significant evidence of immunoreactivity specific to diabetes, thyroid, and gastric-parietal cells in obese patients with NGT. The relatively higher frequency of the diabetes-related IA-2(256-760) autoantibodies in obese patients with NGT may suggest that this autoantibody could be associated with obesity the presence of obesity itself.

No effects of oral vitamin D supplementation on non-alcoholic fatty liver disease in patients with type 2 diabetes: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common hepatic disorder worldwide, reaching prevalence up to 90 % in obese patients with type 2 diabetes (T2D), and representing an independent risk factor for cardiovascular mortality. Furthermore, the coexistence of T2D and NAFLD leads to higher incidence of diabetes' complications and additive detrimental liver outcomes. The existence of a close association between NAFLD and hypovitaminosis D, along with the anti-inflammatory and insulin-sensitizing properties of vitamin D, have been largely described, but vitamin D effects on hepatic fat content have never been tested in a randomized controlled trial. We assessed the efficacy and safety of 24-week oral high-dose vitamin D supplementation in T2D patients with NAFLD. METHODS: This randomized, double-blind, placebo-controlled trial was carried out at the Diabetes Centre of Sapienza University, Rome, Italy, to assess oral treatment with cholecalciferol (2000 IU/day) or placebo in T2D patients with NAFLD. The primary endpoint was reduction of hepatic fat fraction (HFF) measured by magnetic resonance; as hepatic outcomes, we also investigated changes in serum transaminases, CK18-M30, N-terminal Procollagen III Propeptide (P3NP) levels, and Fatty Liver Index (FLI). Secondary endpoints were improvement in metabolic (fasting glycaemia, HbA1c, lipids, HOMA-IR, HOMA-ß, ADIPO-IR, body fat distribution) and cardiovascular (ankle-brachial index, intima-media thickness, flow-mediated dilatation) parameters from baseline to end of treatment. RESULTS: Sixty-five patients were randomized, 26 (cholecalciferol) and 29 (placebo) subjects completed the study. 25(OH) vitamin D significantly increased in the active treated group (48.15 ± 23.7 to 89.80 ± 23.6 nmol/L, P < 0.001); however, no group differences were found in HFF, transaminases, CK18-M30, P3NP levels or FLI after 24 weeks. Vitamin D neither changed the metabolic profile nor the cardiovascular parameters. CONCLUSIONS: Oral high-dose vitamin D supplementation over 24 weeks did not improve hepatic steatosis or metabolic/cardiovascular parameters in T2D patients with NAFLD. Studies with a longer intervention period are warranted for exploring the effect of long time exposure to vitamin D. TRIAL REGISTRATION: This trial was approved on July 2011 by the Ethics Committee of Policlinico Umberto I, Sapienza University of Rome, Italy, and registered at www.clinicaltrialsregister.eu number 2011-003010-17.

Antibodies to post-translationally modified insulin in type 1 diabetes.

AIM/HYPOTHESIS: Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. METHODS: oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). RESULTS: MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. CONCLUSION/INTERPRETATION: These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.

Determination of 21-hydroxylase autoantibodies: inter-laboratory concordance in the Euradrenal International Serum Exchange Program.

BACKGROUND: 21-Hydroxylase autoantibodies (21OHAb) are markers of an adrenal autoimmune process that identifies individuals with autoimmune Addison's disease (AAD). Quality and inter-laboratory agreement of various 21OHAb tests are incompletely known. The objective of the study was to determine inter-laboratory concordance for 21OHAb determinations. METHODS: Sixty-nine sera from 51 patients with AAD and 51 sera from 51 healthy subjects were blindly coded by a randomization center and distributed to 14 laboratories that determined 21OHAb, either by an "in-house" assay (n=9) using in vitro-translated (35)S-21OH or luciferase-labeled 21OH or a commercial kit with (125)I-21OH (n=5). Main outcome measures were diagnostic accuracy of each participating laboratory and inter-laboratory agreement of 21OHAb assays. RESULTS: Intra-assay coefficient of variation ranged from 2.6% to 5.3% for laboratories using the commercial kit and from 5.1% to 23% for laboratories using "in-house" assays. Diagnostic accuracy, expressed as area under ROC curve (AUC), varied from 0.625 to 0.947 with the commercial kit and from 0.562 to 0.978 with "in-house" methods. Cohen's κ of inter-rater agreement was 0.603 among all 14 laboratories, 0.691 among "in-house" laboratories, and 0.502 among commercial kit users. Optimized cutoff levels, calculated on the basis of AUCs, increased the diagnostic accuracy of every laboratory (AUC >0.9 for 11/14 laboratories) and increased the Cohen's κ of inter-rater agreement. Discrepancies in quantitation of 21OHAb levels among different laboratories increased with increasing autoantibody levels. CONCLUSIONS: The quality of 21OHAb analytical procedures is mainly influenced by selection of cutoff value and correct handling of assay materials. A standardization program is needed to identify common standard sera and common measuring units.

An electrochemical immunoassay for the screening of celiac disease in saliva samples.

A highly sensitive electrochemical immunoassay for the initial diagnosis of celiac disease (CD) in saliva samples that overcomes the problems related to its high viscosity and to the low concentration of anti-transglutaminase antigen (tTG) IgA in this medium has been developed for the first time. The system uses magnetic beads (MBs) covered with tTG, which reacts with the anti-tTG IgA antibodies present in positive saliva samples. An anti-human IgA, conjugated with alkaline phosphate (AP) enzyme, was used as the label and a strip of eight magnetized screen-printed electrodes as the electrochemical transducer. In particular, two different immunoassay approaches were optimized and blindly compared to analyze a large number of saliva samples, whose anti-tTG IgA levels were independently determined by the radioimmunoassay (RIA) method. The obtained results, expressed as Ab index, were used to perform a diagnostic test evaluation through the construction of receiver operating characteristic (ROC) curves. The approach, involving a pre-incubation between the anti-human IgA-AP and saliva samples prior to the addition of MBs-tTG, showed a cutoff of 0.022 with 95% clinical sensitivity and 96% clinical specificity. The area under the ROC curve is equal to 1, a result that classifies our test as "perfect." This study demonstrates that it is possible to perform the screening of CD with a rapid, simple, inexpensive, and sensitive method able to detect anti-tTG antibodies in saliva samples, which are easily obtained by non-invasive techniques. This aspect is of fundamental importance to screen a large number of subjects, especially in the pediatric age.

Anti-transglutaminase immunoreactivity and histological lesions of the duodenum in coeliac patients.

Coeliac disease (CD) is characterized by several markers, including anti-transglutaminase auto-antibodies (tTGAb) directed against multiple epitopes of the gliadin protein. We aimed to investigate the correlation among CD duodenal lesions, tTGAb titres and the immunoreactivity against tTG constructs. A total of 345 CD patients (209 females, 136 males, overall median age: 7.3 years) were tested for full-length (fl) tTGAb with a fluid-phase radioimmunoassay. Out of the total, 231 patients were also tested for immunoreactivity against tTG fragments (F1: a.a. 227-687 and F2: a.a. 473-687). Patients were classified according to diffuse (D), patchy (P) or bulb (B) histological lesions. All sera were found fltTGAb positive. Patients with D, P and B lesions had a mean Ab index of 0.84±0.39, 0.57±0.39 and 0.45±0.24, respectively. Mean tTGAb titre varied between D and localized (P+B) patients (0.84±0.39 versus 0.52±0.34, P < 0.0001). Overall, 86.1% of patients were F1 auto-antibody (F1Ab) positive (D: 89%, P: 75%, B: 40%; D versus P+B: P = 0.004) and 49% of patients were F2 auto-antibody (F2Ab) positive (D: 53%, P: 19%, B: 10%; D versus P+B: P = 0.0006). Of the D patients 50.7% showed combined F1Ab-F2Ab (D versus P+B: P = 0.001), whereas 60% of B patients were negative for both F1Ab and F2Ab (B versus D: P < 0.0001). Coeliac-specific tTGAb immunoreactivity correlates with the grading and extension of histological duodenal lesions in CD patients at diagnosis. The immunoreactivity against single and combined tTG fragments is significantly higher in patients with D lesions. This is the first evidence of a distinct coeliac-specific immunoreactivity in patients with different duodenal involvement.

The celiac iceberg: characterization of the disease in primary schoolchildren.

OBJECTIVE: Celiac disease (CD) has a prevalence of 0.55% to 1% in Italy. Identifying CD in schoolchildren to characterize CD iceberg and evaluate the effect of diagnosis in screening-detected children. METHODS: A total of 7377 5- to 8-year-old children were invited to participate. A total of 5733 salivary samples were collected and tested for anti-transglutaminase antibodies (tTGAb), using a fluid-phase radioimmunoassay. Salivary tTGAb-positive children were analyzed for serum antibodies (anti-endomysium antibodies, radioimmunoassay, and enzyme-linked immunosorbent assay tTGAb). Positive children underwent endoscopy and then started gluten-free diet (GFD) and periodical follow-up. RESULTS: Forty-six subjects were found salivary tTGAb-positive and 16 border-line. Forty-five of 46 and 5 of 15 of them were also serum antibody-positive. Forty-two children showed duodenal villous atrophy and 1 had only type 1 lesions. Three children started GFD without performing endoscopy. CD prevalence (including 23 previously diagnosed children with CD) was 1.2%. Considering all 65 celiacs in our sample, a silent CD was found in 64%, typical in 28%, atypical in 7%, and potential in 1%. All patients showed strict adherence to GFD, weight and stature increase, and well-being improvement. Eighty-five percent and all but 2 screening-detected children with CD had Italian parents. CONCLUSIONS: Our sample size, representative of primary schoolchildren of our region, demonstrated that CD prevalence is growing in Italy, with a modified clinical spectrum and iceberg deepness.

Investigating the Relationship Between COVID-19 and Celiac Disease. A Dual Research Approach.

Background: Most evidence on the coronavirus disease 2019 (COVID-19) pandemic, has been obtained from web- or telephone-based surveys. In particular, few laboratory data, often incomplete, have been reported on the frequency of COVID-19-related serology at celiac disease (CD) diagnosis or on the effects of COVID-19 on the development of CD-specific autoimmunity. Objectives: The objective of this retrospective cross-sectional case/control study was to: (1) evaluate the frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in 78 children and adolescents at CD diagnosis (CD, 44 females, median age 7.4 years); (2) evaluate the frequency of IgA-anti-transglutaminase antibodies (IgA-tTGAbs) in 97 nonceliac patients (50 females, median age 9.0 years) who contracted SARS-CoV-2 infection during the pandemic (February-April 2021). As a control (CTRL) group, we analyzed 141 healthy subjects (79 females, median age 9.8 years) enrolled during the pandemic. Methods: SARS-CoV-2 IgM- and IgG-antibodies were detected by chemiluminescent microparticle immunoassays. IgA-tTGAbs were detected by a fluid-phase radioimmunoassay. Results: Six out of 78 (7.7%) CD patients tested positive for SARS-CoV-2Abs, with a frequency not significantly different from CTRL subjects (9.2%). None of the 97 nonceliac COVID-19 patients tested positive for IgA-tTG antibodies. Conclusion: These 2 distinct research approaches showed (1) similar frequencies of SARS-CoV-2 immunoreactivities in CD patients and CTRL subjects and, (2) no ability of SARS-CoV-2 to induce a CD-specific immune response, at least in the 3-4 months following SARS-CoV-2 infection.

No effects of COVID-19 on the development of type 1 diabetes autoimmunity and no evidence of an increased frequency of SARS-CoV-2 antibodies in newly diagnosed type 1 diabetes patients relative to healthy subjects.

AIMS: To evaluate the relationship between SARS-CoV-2 infection and autoimmunity in type 1 diabetes (T1D) and SARS-CoV-2 antibodies frequency at diagnosis of T1D during pandemic. METHODS: The presence of T1D-specific autoimmunity was evaluated in a cohort of 99 children and adolescents without diabetes that contracted SARS-CoV-2 infection. Moreover, the frequency of IgM- and IgG-SARS-CoV-2 antibodies was evaluated in 41 newly diagnosed T1D patients not yet vaccinated against SARS-CoV-2 disease, collected during the pandemic, compared to healthy subjects (CTRL). RESULTS: None of the 99 patients that contracted SARS-CoV-2 infection during the pandemic period was found positive for T1D autoantibodies. The frequency of SARS-CoV-2 antibodies was not significantly different in patients newly diagnosed with T1D (12.2%), compared with CTRL (8.4%). Among SARS-CoV-2 antibody positive T1D patients, 80% were target of diabetes autoantibodies and 60% had another concomitant autoimmune disease. Among the CTRL subjects positive for SARS-CoV-2Abs (n = 10), none was found positive for T1D autoantibodies. CONCLUSIONS: The results of the present study do not confirm, at least in the short term, a role of COVID-19 as a potential trigger of T1D autoimmunity and do not provide evidence of an increased frequency of SARS-CoV-2 antibodies in newly diagnosed T1D patients in comparison with healthy population.

Endoscopic and histological gastric lesions in children with celiac disease: mucosal involvement is not only confined to the duodenum.

OBJECTIVES: Lymphocytic gastritis (LG) has been reported in patients with celiac disease (CD). The aim of the present study was to evaluate gastric mucosa involvement in celiac children and gastroenterological controls (GC). METHODS: In a retrospective study on 226 patients with CD (82  M; median age: 5.7 years) at diagnosis and 154 GC (66  M; median age: 7.4 years), the evaluation of gastric and duodenal mucosa was performed. CD was diagnosed according to the North America Society for Pediatric Gastroenterology, Hepatology, and Nutrition criteria. Gastric lesions were classified according to Updated Sydney System. Anti-gastric parietal cell antibodies (GPCA) were assayed by enzyme-linked immunosorbent assay. RESULTS: A total of 21.2% and 7% of patients with CD showed chronic superficial gastritis (CSG) and LG, respectively. Helicobacter pylori (Hp) infection was found in 6 (2.7%) children with CD (66.7% had CSG, 16.7% LG, and 16.7% interstitial gastritis). CSG was present in 21.4% of controls. No control subject showed LG. Hp infection was found in 24 (15.6%) children with GC (91.7% had CSG). Among patients with CSG, Hp infection was more frequent in controls than in celiac children (P < 0.0001). Ten of 90 patients with CD and 1 of 29 controls were positive for GPCA. CONCLUSIONS: Gastritis is a common finding in children with CD and adolescents. In celiac subjects, CSG is the most frequently detected. Our data suggest the hypothesis that LG may be related to a longer exposure to gluten. The presence of GPCA may suggest the presence of an underlying autoimmune process.

Immunoreactivities Against Different Tyrosine-Phosphatase 2 (IA-2)(256-760) Protein Domains Characterize Distinct Phenotypes in Subjects With LADA.

Antibodies (Abs) against intracellular epitopes of the tyrosine-phosphatase 2 (IA-2) are detected in type 1 diabetes. Abs directed against the IA-2(256-760) portion, with both intra- and extracellular epitopes, are present in people with latent autoimmune diabetes in adults (LADA) and in obese subjects with normal glucose tolerance (NGT). We aim to characterize distribution and clinical features of intra- and extra-cellular IA-2(256-760) immunoreactivities in people with LADA compared to obese people with NGT. The intracellular immunoreactivity represented by immune response against two intracellular IA-2 constructs (IA-2JM(601-630) and IA-2IC(605-979)) was analyzed and related to clinical and biochemical features in 101 people with LADA and in 20 NGT obese subjects, all testing positive for IA-2(256-760) Abs. IA-2 intracellular immunoreactivity showed a frequency of 40.6% in LADA while it was not detected among NGT obese (p<0.001). Amongst LADA, the presence of immunoreactivity against the IA-2 intracellular domains was associated with lower BMI, waist circumference, higher HDL cholesterol and lower triglycerides, lower prevalence of hypertension and higher prevalence of other autoimmune disorders. Immunoreactivity against IA-2 does not involve intracellular domains in the majority of LADA and in obese people with NGT. This study shows that there is heterogeneity in the IA-2 epitopes, associated with different clinical features.

Skeletal status in children and adolescents with new-onset type 1 diabetes: a preliminary study based on bone densitometry and quantitative ultrasound.

INTRODUCTION: Type 1 diabetes (T1D) represents a risk factor for bone loss and impaired bone quality. MATERIAL AND METHODS: We conducted an exploratory retrospective cross-sectional study involving youths with new-onset T1D, to investigate the relationship between lumbar spine dual-energy X-ray absorptiometry (DXA) and phalangeal quantitative ultrasound (QUS) measurements, along with their correlation with markers of bone turnover, glucose homeostasis, and residual ß-cell function. RESULTS: 17 children and adolescents (8 females) with recent-onset T1D were enrolled into this study. Lumbar spine areal bone mineral density (aBMD) and age-adjusted amplitude-dependent speed of sound (AD-SoS) Z-scores were indicative of low BMD status (≤ -2.0 SD) in 11.7% and 17.6% of participants, respectively. Spearman's correlation analysis revealed significant inverse correlations between AD-SoS values and circulating levels of ß-CrossLaps, alkaline phosphatase, and osteocalcin, along with a significant positive correlation between bone transmission time (BTT) values and fasting plasma C-peptide (FCP) levels. There was no statistically significant correlation between DXA-QUS parameters, fasting plasma glucose (FPG), and glycated haemoglobin (HbA1c). Finally, there was a significant positive correlation between lumbar spine aBMD and BTT values. CONCLUSIONS: Our study suggests that DXA and/or QUS parameters may be altered in a small proportion of T1D children and adolescents at the disease onset. Additionally, residual ß-cell function may represent a protective factor against T1D-related detrimental skeletal changes. Large and long-term prospective studies are needed to confirm these preliminary findings since the present study is limited by the retrospective cross-sectional design and by its small sample size.

Measuring adrenal autoantibody response: interlaboratory concordance in the first international serum exchange for the determination of 21-hydroxylase autoantibodies.

21-hydroxylase autoantibodies (21OHAb) are the gold standard immune marker to identify patients with clinical or subclinical autoimmune Addison's disease (AAD). No assessment of interlaboratory concordance has been made for 21OHAb measurement. Serum samples from 267 patients with primary adrenal insufficiency and from 83 healthy control subjects were distributed to four independent laboratories that determined presence and titer of 21OHAb, by using radiobinding assays with either in vitro translated 35S-radiolabelled or 125I-radiolabelled autoantigen. Cohen's κ of inter-rater agreement ranged from 0.857 to 0.983, showing a very good concordance of the positive/negative score among the four laboratories. Passing-Bablok regression showed a good agreement of 21OHAb titers arranged by ranks, but important discrepancies emerged at the Bland-Altman plot, as the repeatability coefficient was much higher than the laboratory cut-offs, which indicates that results from different laboratories cannot be used interchangeably. A standardization international program for 21OHAb measurement is strongly needed.

First salivary screening of celiac disease by detection of anti-transglutaminase autoantibody radioimmunoassay in 5000 Italian primary schoolchildren.

OBJECTIVE: The high prevalence of celiac disease (CD) prompted us to evaluate a new, noninvasive disease screening strategy. The aim was to identify CD in 6- to 8-year-old children for a timely diagnosis, start gluten-free diet (GFD) in compliant subjects, achieve the growth target, and prevent CD complications. METHODS: Five thousand subjects were invited to participate in the study. Four thousand forty-eight saliva samples were tested for anti-tissue transglutaminase (tTG) immunoglobulin (Ig)A using a fluid-phase radioimmunoprecipitation method. Positive children were tested for serum radioimmunoassay tTG IgA, enzyme-linked immunosorbent assay tTG IgA, and anti-endomysium IgA. Children confirmed as positive by serum assays underwent endoscopy with duodenal biopsies and, at the diagnosis of CD, were suggested to start GFD. RESULTS: Consent was obtained from 4242 parents (84.8%) for the screening to be performed, and adequate saliva samples were collected from 4048 children (95.4%). Thirty-two children were found to be salivary tTG IgA positive and 9 with borderline autoantibody levels. Thirty-one of the 32 and 3 of the 9 subjects were also serum positive. Twenty-eight children showed villous atrophy when undergoing intestinal biopsy, whereas 1 had Marsh 1 lesions; 3 children were suggested to start GFD without performing endoscopy. CD prevalence in the population investigated (including 19 CD known cases) was 1.16%. The ratio between screening-detected patients and those diagnosed before the screening was 3:2. The ratio between symptomatic and asymptomatic patients was 1:1.6. CONCLUSIONS: We demonstrated that it is possible to perform a powerful, simple, well-accepted, and sensitive CD screening using saliva. Until now, the compliance with GFD in children with CD has been optimal.

Fasting Neurotensin Levels in Pediatric Celiac Disease Compared with a Control Cohort.

BACKGROUND AND AIMS: Neurotensin (NT) is a gut hormone secreted by specific endocrine cells scattered throughout the epithelial layer of the small intestine, which has been identified as an important mediator in several gastrointestinal functions and disease conditions. Its potential involvement in celiac disease (CD) has been investigated, but there are conflicting findings. The aim of this study was to evaluate serum NT levels in children with CD at diagnosis, compared to a control group, and to investigate whether NT correlated in CD patients with symptoms, antibody response, and intestinal mucosal damage. Materials and Methods. Children (1-16 years old) undergoing gastrointestinal endoscopy for CD or for other clinical reasons were included in this study. Patients with CD diagnosed according to the 2012 European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines without biopsy were also recruited. Fasting serum samples were analyzed for NT levels using ELISA. Logistic regression, Wilcoxon rank sum, and Spearman's rank tests were used for statistical analysis. RESULTS: Thirty children (18 females, 2.2-15.9 years old) were enrolled. Of 25 patients who underwent endoscopy, 9 were CD patients, 13 were controls, and 3 were excluded due to nonspecific inflammation at duodenal biopsy. CD was diagnosed in 5 patients without biopsy. NT median was higher in CD patients compared to controls (13.25 (IQR 9.4-17.5) pg/ml vs. 7.8 (IQR 7.6-10) pg/ml; p = 0.02). No statistically significant association between NT and clinical, serological, or histological data of CD was observed in this CD cohort. CONCLUSIONS: To our knowledge, this is the first study that evaluates NT in CD children from Italy. Results show that NT is higher in the serum of CD children at diagnosis compared to controls. However, larger-scale studies are required to validate these findings. Whether serum NT levels can be an adjunctive marker for pediatric CD remains currently elusive.

GAD and IA-2 autoantibody detection in type 1 diabetic patient saliva.

Some attempts have been made in assaying glutamic-acid decarboxylase autoantibodies (GADA) in type 1 diabetic patient (T1DM) saliva. However, these salivary assays did not show sufficient sensitivity and specificity in comparison to serum assays. In this study we evaluated the ability of a fluid-phase (35)S-radioimmunoassay to detect GADA and tyrosine phosphatase 2 autoantibodies (IA-2A) in 70 T1DM, 24 T1DM first degree relatives (FDR) and 76 healthy subject saliva. Paired saliva and serum samples were collected from each subject and analyzed. GADA were detected in 45/70 (64.3%) sera and 43/70 (61.4%) T1DM saliva, respectively. IA-2A were detected in 33/70 (47.1%) sera and 30/70 (42.9%) T1DM saliva, respectively. All FDR serum/saliva samples were autoantibody negative. In conclusion, we here report that GADA and IA-2A are detectable with high sensitivity and specificity in human saliva, a specimen which can be easily collected by non-invasive procedures and may represent a reliable tool for the study of T1DM autoimmunity.

A report on the International Transglutaminase Autoantibody Workshop for Celiac Disease.

OBJECTIVES: Measurement of transglutaminase autoantibodies (TGAA) is considered to be the most efficient single serologic test for celiac disease (CD) by the American Gastroenterological Association Institute. We hypothesized that a large international collaborative effort toward improving and standardizing TGAA measurement is both feasible and necessary. The primary aim of this workshop is to compare TGAA assays among various research and clinical laboratories to examine assay concordance and improve (and eventually standardize) the TGAA assay. METHODS: A total of 20 laboratories (5 commercial laboratories, 15 research and clinical laboratories) participated that included enzyme-linked immunosorbent assay (ELISA) and radiobinding assays. A total of 150 serum samples were distributed to each laboratory, with each laboratory receiving an equal aliquot that was coded and blinded, composed of 100 healthy control sera and 50 CD sera. RESULTS: Laboratory sensitivity ranged from 69% to 93% and specificity ranged from 96% to 100%. By receiver operator characteristic analysis, the area under the curve (C index) ranged from 0.9488 to 0.9904. When analyzing for linear correlation, r-squared was as high as 0.8882 but as low as 0.4244 for the celiac samples between different laboratories performing ELISA. CONCLUSIONS: This transglutaminase autoantibody workshop allows for larger-scale international participation for the purposes of improving and eventually standardizing the TGAA assay with subsequent workshops.