Islet cell stress induced by insulin-degrading enzyme deficiency promotes regeneration and protection from autoimmune diabetes.

Tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with variable demand for insulin. Here, we asked how insulin-degrading enzyme (IDE) affects beta cell adaptation to metabolic and immune stress. C57BL/6 and autoimmune non-obese diabetic (NOD) mice lacking IDE were exposed to proteotoxic, metabolic, and immune stress. IDE deficiency induced a low-level UPR with islet hypertrophy at the steady state, rapamycin-sensitive beta cell proliferation enhanced by proteotoxic stress, and beta cell decompensation upon high-fat feeding. IDE deficiency also enhanced the UPR triggered by proteotoxic stress in human EndoC-ßH1 cells. In Ide-/- NOD mice, islet inflammation specifically induced regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. These findings establish a role of IDE in islet cell protein homeostasis, demonstrate how its absence induces metabolic decompensation despite beta cell proliferation, and UPR-independent islet regeneration in the presence of inflammation.

Real-life data comparing the efficacy of vigabatrin and oral steroids given sequentially or combined for infantile epileptic spasms syndrome.

AIMS: The prognosis of Infantile epileptic spasm syndrome (IESS), relates to the underlying etiology and delay in controlling epileptic spasms. Based on the spasm-free rate, a randomized controlled trial has demonstrated the superiority of combining oral steroids and vigabatrin over oral steroids alone but confirmation in real-life conditions is mandatory. METHODS: We compared two real-life IESS cohorts: a multicenter, retrospective cohort of 40 infants treated with vigabatrin followed by a sequential (ST) addition of steroids, and a prospective, single-center cohort of 58 infants treated with an immediate combination of vigabatrin and steroids (CT). RESULTS: The two cohorts were similar. When the rate of spasm-free infants in the two cohorts was compared on day 14, a significant difference was observed between the ST (27,5 %) and CT cohorts (64 %) (p < 0.0004). This difference remained significant on day 30, with 55 % spasm-free patients in the ST cohort compared to 76 % in the CT cohort (p = 0.03). After the infants had received both vigabatrin and steroids, without taking into account the time point after treatment initiation, no significant difference was observed in the spasm-free rate between the two cohorts (p = 0.38). INTERPRETATION: Real-life data confirm the interest of combination therapy as a first-line treatment for IESS.

Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes.

Appropriate tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with highly variable demand for insulin synthesis. An efficient UPR ensures a sufficient beta cell mass and secretory output but can also affect beta cell resilience to autoimmune aggression. The factors regulating protein homeostasis in the face of metabolic and immune challenges are insufficiently understood. We examined beta cell adaptation to stress in mice deficient for insulin-degrading enzyme (IDE), a ubiquitous protease with high affinity for insulin and genetic association with type 2 diabetes. IDE deficiency induced a low-level UPR in both C57BL/6 and autoimmune non-obese diabetic (NOD) mice, associated with rapamycin-sensitive beta cell proliferation strongly enhanced by proteotoxic stress. Moreover, in NOD mice, IDE deficiency protected from spontaneous diabetes and triggered an additional independent pathway, conditional on the presence of islet inflammation but inhibited by proteotoxic stress, highlighted by strong upregulation of regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. Our findings establish a key role of IDE in islet cell protein homeostasis, identify a link between low-level UPR and proliferation, and reveal an UPR-independent anti-inflammatory islet cell response uncovered in the absence of IDE of potential interest in autoimmune diabetes.

Cross-presentation by the others.

The critical role of conventional dendritic cells in physiological cross-priming of immune responses to tumors and pathogens is widely documented and beyond doubt. However, there is ample evidence that a wide range of other cell types can also acquire the capacity to cross-present. These include not only other myeloid cells such as plasmacytoid dendritic cells, macrophages and neutrophils, but also lymphoid populations, endothelial and epithelial cells and stromal cells including fibroblasts. The aim of this review is to provide an overview of the relevant literature that analyzes each report cited for the antigens and readouts used, mechanistic insight and in vivo experimentation addressing physiological relevance. As this analysis shows, many reports rely on the exceptionally sensitive recognition of an ovalbumin peptide by a transgenic T cell receptor, with results that therefore cannot always be extrapolated to physiological settings. Mechanistic studies remain basic in most cases but reveal that the cytosolic pathway is dominant across many cell types, while vacuolar processing is most encountered in macrophages. Studies addressing physiological relevance rigorously remain exceptional but suggest that cross-presentation by non-dendritic cells may have significant impact in anti-tumor immunity and autoimmunity.

Compromised mitochondrial quality control triggers lipin1-related rhabdomyolysis.

LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo.

Cross-sectional case-control study of the relationships between pharyngeal compliance and heart rate variability indices in childhood obstructive sleep apnoea.

A combination of noradrenergic and antimuscarinic agents reduces the apnea-hypopnea index (AHI) in adult patients with obstructive sleep apnoea (OSA) via reduced upper airway collapsibility, suggesting that a shift in the sympathovagal balance improves OSA. The objectives of our present case-control study were to assess heart rate variability (HRV) indices in the stages of sleep in children with and without OSA to evaluate OSA-induced sleep HRV modifications and to assess whether increased collapsibility measured during wakefulness is associated with reduced sympathetic activity during non-rapid eye movement (NREM) sleep. Three groups of 15 children were matched by sex, age, z-score of body mass index and ethnicity: non-OSA (obstructive AHI [OAHI] <2 events/hr), mild (OAHI ≥2 to <5 events/hr) or moderate-severe (OAHI ≥5 events/hr) OSA. Pharyngeal compliance was measured during wakefulness using acoustic pharyngometry. HRV indices (time and frequency domain variables) were calculated on 5-min electrocardiography recordings from polysomnography during wakefulness, NREM and REM sleep in periods free of any event. As compared to children without OSA, those with OSA (n = 30) were characterised by increased compliance and no physiological parasympathetic tone increase in REM sleep. Children with increased pharyngeal compliance (n = 21) had a higher OAHI due to higher AHI in NREM sleep, whereas their sympathetic tone was lower than that of those with normal compliance (n = 24). In conclusion, children with increased pharyngeal compliance exhibit decreased sympathetic tone associated with increased AHI in NREM sleep. Therapeutics directed at sympathovagal balance modifications should be tested in childhood OSA.

Kinesin-1 regulates antigen cross-presentation through the scission of tubulations from early endosomes in dendritic cells.

Dendritic cells (DCs) constitute a specialized population of immune cells that present exogenous antigen (Ag) on major histocompatibility complex (MHC) class I molecules to initiate CD8 + T cell responses against pathogens and tumours. Although cross-presentation depends critically on the trafficking of Ag-containing intracellular vesicular compartments, the molecular machinery that regulates vesicular transport is incompletely understood. Here, we demonstrate that mice lacking Kif5b (the heavy chain of kinesin-1) in their DCs exhibit a major impairment in cross-presentation and thus a poor in vivo anti-tumour response. We find that kinesin-1 critically regulates antigen cross-presentation in DCs, by controlling Ag degradation, the endosomal pH, and MHC-I recycling. Mechanistically, kinesin-1 appears to regulate early endosome maturation by allowing the scission of endosomal tubulations. Our results highlight kinesin-1's role as a molecular checkpoint that modulates the balance between antigen degradation and cross-presentation.

IRAP Endosomes Control Phagosomal Maturation in Dendritic Cells.

Dendritic cells (DCs) contribute to the immune surveillance by sampling their environment through phagocytosis and endocytosis. We have previously reported that, rapidly following uptake of extracellular antigen into phagosomes or endosomes in DCs, a specialized population of storage endosomes marked by Rab14 and insulin-regulated aminopeptidase (IRAP) is recruited to the nascent antigen-containing compartment, thereby regulating its maturation and ultimately antigen cross-presentation to CD8+ T lymphocytes. Here, using IRAP-/- DCs, we explored how IRAP modulates phagosome maturation dynamics and cross-presentation. We find that in the absence of IRAP, phagosomes acquire more rapidly late endosomal markers, are more degradative, and show increased microbicidal activity. We also report evidence for a role of vesicle trafficking from the endoplasmic reticulum (ER)-Golgi intermediate compartment to endosomes for the formation or stability of the IRAP compartment. Moreover, we dissect the dual role of IRAP as a trimming peptidase and a critical constituent of endosome stability. Experiments using a protease-dead IRAP mutant and pharmacological IRAP inhibition suggest that IRAP expression but not proteolytic activity is required for the formation of storage endosomes and for DC-typical phagosome maturation, whereas proteolysis is required for fully efficient cross-presentation. These findings identify IRAP as a key factor in cross-presentation, trimming peptides to fit the major histocompatibility complex class-I binding site while preventing their destruction through premature phagosome maturation.

Innate Immune Signals Induce Anterograde Endosome Transport Promoting MHC Class I Cross-Presentation.

Both cross-presentation of antigens by dendritic cells, a key pathway triggering T cell immunity and immune tolerance, and survival of several pathogens residing in intracellular vacuoles are intimately linked to delayed maturation of vesicles containing internalized antigens and microbes. However, how early endosome or phagosome identity is maintained is incompletely understood. We show that Toll-like receptor 4 (TLR4) and Fc receptor ligation induces interaction of the GTPase Rab14 with the kinesin KIF16b mediating plus-end-directed microtubule transport of endosomes. As a result, Rab14 recruitment to phagosomes delays their maturation and killing of an internalized pathogen. Enhancing anterograde transport by overexpressing Rab14, promoting the GTP-bound Rab14 state, or inhibiting retrograde transport upregulates cross-presentation. Conversely, reducing Rab14 expression, destabilizing Rab14 endosomes, and inhibiting anterograde microtubule transport by Kif16b knockdown compromise cross-presentation. Therefore, regulation of early endosome trafficking by innate immune signals is a critical parameter in cross-presentation by dendritic cells.

Cardiac function and exercise adaptation in 8 children with LPIN1 mutations.

INTRODUCTION: Lipin-1 deficiency is a major cause of rhabdomyolysis that are precipitated by febrile illness. The prognosis is poor, with one-third of patients dying from cardiac arrest during a crisis episode. Apart from acute rhabdomyolysis, most patients are healthy, showing normal clinical and cardiac ultrasound parameters. PATIENTS AND METHODS: We report cardiac and exercise examinations of 8 children carrying two LPIN1 mutations. The examinations were performed outside of a myolysis episode, but one patient presented with fever during one examination. RESULTS: All but one patient displayed normal resting cardiac function, as determined by echocardiography. One patient exhibited slight left ventricular dysfunction at rest and a lack of increased stroke volume during cycle ramp exercise. During exercise, peripheral muscle adaptation was impaired in 2 patients compared to healthy controls: they presented an abnormal increase in cardiac output relative to oxygen uptake: dQ/dVO2=8.2 and 9.5 (>2DS of controls population). One patient underwent 2 exercise tests; during one test, the patient was febrile, leading to acute rhabdomyolysis in the following hours. He exhibited changes in recovery muscle reoxygenation parameters and an increased dQ/dVO2 during exercise compared with that under normothermia (7.9 vs 6), which did not lead to acute rhabdomyolysis. The four patients assessed by cardiac 1H-magnetic resonance spectroscopy exhibited signs of intracardiac steatosis. CONCLUSION: We observed abnormal haemodynamic profiles during exercise in 3/8 patients with lipin-1 deficiency, suggesting impaired muscle oxidative phosphorylation during exercise. Fever appeared to be an aggravating factor. One patient exhibited moderate cardiac dysfunction, which was possibly related to intracardiac stored lipid toxicity.

IRAP+ endosomes restrict TLR9 activation and signaling.

The retention of intracellular Toll-like receptors (TLRs) in the endoplasmic reticulum prevents their activation under basal conditions. TLR9 is activated by sensing ligands in specific endosomal-lysosomal compartments. Here we identified IRAP+ endosomes as major cellular compartments for the early steps of TLR9 activation in dendritic cells (DCs). Both TLR9 and its ligand, the dinucleotide CpG, were present as cargo in IRAP+ endosomes. In the absence of the aminopeptidase IRAP, the trafficking of CpG and TLR9 to lysosomes and signaling via TLR9 were enhanced in DCs and in mice following bacterial infection. IRAP stabilized CpG-containing endosomes by interacting with the actin-nucleation factor FHOD4, which slowed the trafficking of TLR9 toward lysosomes. Thus, endosomal retention of TLR9 via the interaction of IRAP with the actin cytoskeleton is a mechanism that prevents hyper-activation of TLR9 in DCs.

Optimization and Structure-Activity Relationships of Phosphinic Pseudotripeptide Inhibitors of Aminopeptidases That Generate Antigenic Peptides.

The oxytocinase subfamily of M1 aminopeptidases, consisting of ER aminopeptidase 1 (ERAP1), ER aminopeptidase 2 (ERAP2), and insulin-regulated aminopeptidase (IRAP), plays critical roles in the generation of antigenic peptides and indirectly regulates human adaptive immune responses. We have previously demonstrated that phosphinic pseudotripeptides can constitute potent inhibitors of this group of enzymes. In this study, we used synthetic methodologies able to furnish a series of stereochemically defined phosphinic pseudotripeptides and demonstrate that side chains at P1' and P2' positions are critical determinants in driving potency and selectivity. We identified low nanomolar inhibitors of ERAP2 and IRAP that display selectivity of more than 2 and 3 orders of magnitude, respectively. Cellular analysis demonstrated that one of the compounds that is a selective IRAP inhibitor can reduce IRAP-dependent but not ERAP1-dependent cross-presentation by dendritic cells with nanomolar efficacy. Our results encourage further preclinical development of phosphinic pseudotripeptides as regulators of adaptive immune responses.

Screening Identifies Thimerosal as a Selective Inhibitor of Endoplasmic Reticulum Aminopeptidase 1.

We employed virtual screening followed by in vitro evaluation to discover novel inhibitors of ER aminopeptidase 1, an important enzyme for the human adaptive immune response that has emerged as an attractive target for cancer immunotherapy and the control of autoimmunity. Screening hits included three structurally related compounds carrying the (E)-N'-((1H-indol-3-yl)methylene)-1H-pyrazole-5-carbohydrazide scaffold and (2-carboxylatophenyl)sulfanyl-ethylmercury as novel ERAP1 inhibitors. The latter, also known as thimerosal, a common component in vaccines, was found to inhibit ERAP1 in the submicromolar range and to present strong selectivity versus the homologous aminopeptidases ERAP2 and IRAP. Cell-based analysis indicated that thimerosal can effectively reduce ERAP1-dependent cross-presentation by dendritic cells in a dose-dependent manner.

A unique CD8(+) T lymphocyte signature in pediatric type 1 diabetes.

Human type 1 diabetes results from a destructive auto-reactive immune response in which CD8(+) T lymphocytes play a critical role. Given the intense ongoing efforts to develop immune intervention to prevent and/or cure the disease, biomarkers suitable for prediction of disease risk and progress, as well as for monitoring of immunotherapy are required. We undertook separate multi-parameter analyses of single naïve and activated/memory CD8(+) T lymphocytes from pediatric and adult patients, with the objective of identifying cellular profiles associated with onset of type 1 diabetes. We observe global perturbations in gene and protein expression and in the abundance of T cell populations characterizing pediatric but not adult patients, relative to age-matched healthy individuals. Pediatric diabetes is associated with a unique population of CD8(+) T lymphocytes co-expressing effector (perforin, granzyme B) and regulatory (transforming growth factor ß, interleukin-10 receptor) molecules. This population persists after metabolic normalization and is especially abundant in children with high titers of auto-antibodies to glutamic acid decarboxylase and with elevated HbA1c values. These findings highlight striking differences between pediatric and adult type 1 diabetes, indicate prolonged large-scale perturbations in the CD8(+) T cell compartment in the former, and suggest that CD8(+)CD45RA(-) T cells co-expressing effector and regulatory factors are of interest as biomarkers in pediatric type 1 diabetes.

Beta cell antigens in type 1 diabetes: triggers in pathogenesis and therapeutic targets.

Research focusing on type 1 diabetes (T1D) autoantigens aims to explore our understanding of these beta cell proteins in order to design assays for monitoring the pathogenic autoimmune response, as well as safe and efficient therapies preventing or stopping it. In this review, we will discuss progress made in the last 5 years with respect to mechanistic understanding, diagnostic monitoring, and therapeutic modulation of the autoantigen-specific cellular immune response in T1D. Some technical progress in monitoring tools has been made; however, the potential of recent technologies for highly multiplexed exploration of human cellular immune responses remains to be exploited in T1D research, as it may be the key to the identification of surrogate markers of disease progression that are still wanting. Detailed analysis of autoantigen recognition by T cells suggests an important role of non-conventional antigen presentation and processing in beta cell-directed autoimmunity, but the impact of this in human T1D has been little explored. Finally, therapeutic administration of autoantigens to T1D patients has produced disappointing results. The application of novel modes of autoantigen administration, careful translation of mechanistic understanding obtained in preclinical studies and in vitro with human cells, and combination therapies including CD3 antibodies may help to make autoantigen-based immunotherapy for T1D a success story in the future.

Cholestasis Reveals Severe Cortisol Deficiency in Neonatal Pituitary Stalk Interruption Syndrome.

OBJECTIVES: Cholestasis has been reported during the course of congenital hypothalamic-pituitary deficiency, but crucial information is lacking regarding both its origin and prognosis. We aimed to characterize the course of cholestasis and factors contributing to it in patients with deficiency due to pituitary stalk interruption syndrome (PSIS). METHODS: We conducted a retrospective single-center, case-cohort study including 16 patients with PSIS diagnosed before one year of age. We collected clinical and biological parameters from medical records and compared the characteristics of the endocrine syndrome in PSIS patients with and without cholestasis. RESULTS: 5/16 patients had cholestasis, all with a neonatal onset and multiple hypothalamic-pituitary deficiency. Patients with cholestasis presented with lower Apgar score and higher rate of ophthalmic malformations: 3/5 vs 1/11, p = 0.03 and 5/5 vs 4/11, p = 0.02, respectively. The plasma cortisol level was strongly decreased in patients with cholestasis: 12.4 ng/mL (8-15 ng/mL) vs 79.4 ng/mL (10-210 ng/mL), p = 0.04. Cholestasis resolved within 9 months following hormone supplementation. No development of chronic liver disease was observed during a median follow-up of 9.4 years (range, 1.3-13.3 years). CONCLUSIONS: Cholestasis is a frequent symptom at presentation of PSIS during the neonatal period that may help earlier diagnosis and that indicates a profound cortisol deficiency.

Acute rhabdomyolysis and inflammation.

Rhabdomyolysis results from the rapid breakdown of skeletal muscle fibers, which leads to leakage of potentially toxic cellular content into the systemic circulation. Acquired causes by direct injury to the sarcolemma are most frequent. The inherited causes are: i) metabolic with failure of energy production, including mitochondrial fatty acid ß-oxidation defects, LPIN1 mutations, inborn errors of glycogenolysis and glycolysis, more rarely mitochondrial respiratory chain deficiency, purine defects and peroxysomal α-methyl-acyl-CoA-racemase defect (AMACR), ii) structural causes with muscle dystrophies and myopathies, iii) calcium pump disorder with RYR1 gene mutations, iv) inflammatory causes with myositis. Irrespective of the cause of rhabdomyolysis, the pathology follows a common pathway, either by the direct injury to sarcolemma by increased intracellular calcium concentration (acquired causes) or by the failure of energy production (inherited causes), which leads to fiber necrosis. Rhabdomyolysis are frequently precipitated by febrile illness or exercise. These conditions are associated with two events, elevated temperature and high circulating levels of pro-inflammatory mediators such as cytokines and chemokines. To illustrate these points in the context of energy metabolism, protein thermolability and the potential benefits of arginine therapy, we focus on a rare cause of rhabdomyolysis, aldolase A deficiency. In addition, our studies on lipin-1 (LPIN1) deficiency raise the possibility that several diseases involved in rhabdomyolysis implicate pro-inflammatory cytokines and may even represent primarily pro-inflammatory diseases. Thus, not only thermolability of mutant proteins critical for muscle function, but also pro-inflammatory cytokines per se, may lead to metabolic decompensation and rhabdomyolysis.

3,4-diaminobenzoic acid derivatives as inhibitors of the oxytocinase subfamily of M1 aminopeptidases with immune-regulating properties.

Members of the oxytocinase subfamily of M1 aminopeptidases (ERAP1, ERAP2, and IRAP) play important roles in both the adaptive and innate human immune responses. Their enzymatic activity can contribute to the pathogenesis of several major human diseases ranging from viral and parasitic infections to autoimmunity and cancer. We have previously demonstrated that diaminobenzoic acid derivatives show promise as selective inhibitors for this group of aminopeptidases. In this study, we have thoroughly explored a series of 3,4-diaminobenzoic acid derivatives as inhibitors of this class of enzymes, achieving submicromolar inhibitors for ERAP2 (IC50 = 237 nM) and IRAP (IC50 = 105 nM). Cell-based analysis indicated that the lead compounds can be effective in downregulating macrophage activation induced by lipopolysaccharide and interferon-γ as well as cross-presentation by bone marrow-derived dendritic cells. Our results indicate that this class of inhibitors may be useful for the targeted downregulation of immune responses.

No major role for insulin-degrading enzyme in antigen presentation by MHC molecules.

Antigen presentation by MHC class I molecules requires degradation of epitope source proteins in the cytosol. Although the preeminent role of the proteasome is clearly established, evidence suggesting a significant role for proteasome-independent generation of class I ligands has been reported repeatedly. However, an enzyme responsible for such a role has not been identified. Recently insulin-degrading enzyme (IDE) was shown to produce an antigenic peptide derived from the tumor antigen MAGE-A3 in an entirely proteasome-independent manner, raising the question of the global impact of IDE in MHC class I antigen processing. Here we report that IDE knockdown in human cell lines, or knockout in two different mouse strains, has no effect on cell surface expression of various MHC class I molecules, including allomorphs such as HLA-A3 and HLA-B27 suggested to be loaded in an at least a partly proteasome-independent manner. Moreover, reduced or absent IDE expression does not affect presentation of five epitopes including epitopes derived from beta amyloid and proinsulin, two preferred IDE substrates. Thus, IDE does not play a major role in MHC class I antigen processing, confirming the dominant and almost exclusive role of the proteasome in cytosolic production of MHC class I ligands.

Monitoring of anti-transglutaminase autoantibodies in pediatric celiac disease using a sensitive radiobinding assay.

OBJECTIVES: The diagnosis of celiac disease (CD) is based on the histological identification of gluten-sensitive enteropathy and detection of anti-tissue transglutaminase antibodies (tTGA) and/or endomysial antibodies. Serial measurements of tTGA are now recommended as a follow-up strategy to monitor compliance with a gluten-free diet (GFD). We evaluated the performances of a quantitative radiobinding assay (RBA) of tTGA immunoglobulin A at diagnosis and during monitoring of GFD in pediatric CD. METHODS: Eighty children with confirmed CD were selected. Levels of serum tTGA measured by RBA and a commercial enzyme-linked immunosorbent assay (ELISA) were compared at diagnosis. The relation between RBA-tTGA levels and histological damage was analyzed, as well as the time course of tTGA clearance during GFD. RESULTS: Both RBA and ELISA showed high sensitivity and specificity for tTGA detection at diagnosis. There was no relation between RBA-tTGA levels at diagnosis and severity of mucosal damage. Upon initiation of GFD, the rate of RBA-tTGA positivity declined slower than that of endomysial antibodies positivity, with >50% of the children still tTGA positive at year 5; however, tTGA levels decreased rapidly during the first year of GFD and more slowly thereafter. Children who seroreverted had lower tTGA levels at diagnosis (2080±1554 cpm) than those who remained tTGA positive throughout follow-up (3688±1435 cpm). CONCLUSIONS: The high sensitivity of RBA is likely responsible for higher tTGA positivity rates during GFD than previously reported with ELISA. A decreasing trend for tTGA levels may represent a better surrogate marker of compliance with GFD than absolute normal tTGA levels.