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Prolyl endopeptidase-like (PREPL) deficiency (MIM#616224) is a rare congenital disorder characterised by neonatal hypotonia and feeding difficulties, growth hormone (GH) deficiency and hypergonadotropic hypogonadism. This syndrome is an autosomal recessive disease resulting from mutations in the PREPL gene (MIM#609557). Herein we report a 7-year-old female patient with biallelic mutations in PREPL (c.1528C>T in one allele and a whole gene deletion in the other) with early growth impairment in infancy. Growth hormone deficiency was confirmed at 20 months of life. Recombinant growth hormone treatment was introduced with a good response. Her clinical features were similar to those of previously reported cases. The description of new patients with PREPL deficiency syndrome is essential to better delineate the phenotypic and genotypic spectrum of the disease.

Neonatal and infantile cholestasis (NIC) can represent the onset of a surgically correctable disease and of a genetic or metabolic disorder worthy of medical treatment. Timely recognition of NIC and identification of the underlying etiology are paramount to improve outcomes. Upon invitation by the Italian National Institute of Health (ISS), an expert working grouped was formed to formulate evidence-based positions on current knowledge about the diagnosis of NIC. A systematic literature search was conducted to collect evidence about epidemiology, etiology, clinical aspects and accuracy of available diagnostic tests in NIC. Evidence was scored using the GRADE system. All recommendations were approved by a panel of experts upon agreement of at least 75% of the members. The final document was approved by all the panel components. This position document summarizes the collected statements and defines the best-evidence diagnostic approach to cholestasis in the first year of life.

The aim of this study is the evaluation of the safety and the efficacy of long-term combination therapy deferasirox plus desferrioxamine and deferasirox plus deferiprone in a large group of transfusion-dependent thalassemia patients with high values of serum ferritin and/or magnetic resonance, indicative of severe liver and cardiac iron accumulation. Sixteen adults with transfusion-dependent thalassemia were treated simultaneously with deferasirox plus desferrioxamine, while another 42 patients (seven children) were treated with deferasirox plus deferiprone. The hepatic and cardiac iron overload was assessed prior to treatment and then annually with magnetic resonance imaging, and the serum ferritin was measured monthly. Adverse events were checked at each transfusion visit. The safety of both the combinations was consistent with established monotherapies. Both treatments were able to decrease the serum ferritin and liver iron concentration over time, depending on the level of compliance with therapy. Cardiac iron measured as R2* did not significantly change in patients treated with deferasirox plus desferrioxamine. Most patients with MRI indicative of myocardial siderosis at the beginning of treatment reached normal values of cardiac iron at the last determination if treated with deferasirox plus desferrioxamine. The greatest limitation of these therapies was low patient adherence to the two drugs, which is not surprising considering that the need for an intensive chelation is generally linked to previous issues of compliance.

Purpose Thyroid dyshormonogenesis is a heterogeneous group of hereditary diseases produced by a total/partial blockage of the biochemical processes of thyroid-hormone synthesis and secretion. Paired box 8 (PAX8) is essential for thyroid morphogenesis and thyroid hormone synthesis. We aimed to identify PAX8 variants in patients with thyroid dyshormonogenesis and to analyze them with in vitro functional studies. Patients and Methods Nine pediatric patients with a eutopic thyroid gland were analyzed by the Catalan screening program for congenital hypothyroidism. Scintigraphies showed absent, low, or normal uptake. Only one patient had a hypoplastic gland. On reevaluation, perchlorate discharge test was negative or compatible with partial iodine-organization deficit. After evaluation, 8 patients showed permanent mild or severe hypothyroidism. Massive-sequencing techniques were used to detect variants in congenital hypothyroidism-related genes. In vitro functional studies were based on transactivating activity of mutant PAX8 on a TG-gene promoter and analyzed by a dual-luciferase assays. Results We identified 7 heterozygous PAX8 exonic variants and 1 homozygous PAX8 splicing variant in 9 patients with variable phenotypes of thyroid dyshormonogenesis. Five were novel and 5 variants showed a statistically significant impaired transcriptional activity of TG promoter: 51% to 78% vs the wild type. Conclusions Nine patients presented with PAX8 candidate variants. All presented with a eutopic thyroid gland and 7 had deleterious variants. The phenotype of affected patients varies considerably, even within the same family; but, all except the homozygous patient presented with a normal eutopic thyroid gland and thyroid dyshormonogenesis. PAX8 functional studies have shown that 6 PAX8 variants are deleterious. Our studies have proven effective in evaluating these variants.

Background The COVID-19 pandemic and associated public health measures have had a profound impact on health systems worldwide. The aim of this study was to assess quantitative and qualitative changes in Pediatric Emergency Department (PED) visits in Sardinia, Italy, during the early period of the COVID-19 pandemic. Methods We retrospectively investigated the number and characteristics of visits to two major Sardinian PEDs, in the periods January-June 2020 and January-June 2019. Results From January to June 2020, 8399 PED visits with 1160 hospital admissions (13.8% of PED visits) were registered, compared with 15,692 PED visits (Δ = -46.5%) and 1819 hospital admissions (11.6% of PED visits) occurring from January to June 2019. Comparing January-June 2020 with January-June 2019, we found differences in the percentage of visits for age groups, and significant changes in the proportion of triage codes, with a decrease in green codes (72.1% vs 74.2%, respectively) and an increase in white codes (19.0% vs 16.5%, respectively). Moreover, in the period January-June 2020, the frequency of skin disorders and acute respiratory disease significantly decreased, while the frequency of trauma, acute surgical disease, intoxication, and neuropsychiatric disease significantly increased. Conclusions After the beginning of the Italian lockdown, we observed a marked drop in the number of PED visits, an increase in hospital admission rate, and radical changes in the reason for visit.

Ceftriaxone is an antibiotic agent frequently used in paediatric hospital practice for the treatment of severe bacterial infections. The use of this agent can result in cholelithiasis and/or biliary sludge, more commonly in children than in adults. This systematic review was aimed at analysing available literature concerning ceftriaxone-associated biliary pseudolithiasis in paediatric patients, with a special emphasis on the clinical aspects. A literature analysis was performed using Medline and Embase electronic databases (articles published in English up to December 2019), with the search terms and combinations as follows:'ceftriaxone', 'cholelithiasis', 'biliary sludge' 'gallstones' 'neonates' 'children' 'clinical aspects' 'management'. Several case reports, case series and prospective/retrospective studies have documented a relationship between ceftriaxone treatment and biliary pseudolithiasis in the paediatric population, even though literature data regarding neonates and infants are scarce. Ceftriaxone-associated biliary pseudolithiasis is dose-dependent and usually asymptomatic but, sometimes, it may present with abdominal pain, nausea and emesis. Abdominal ultrasonography should be performed when this complication is suspected. Generally, ceftriaxone-associated cholelithiasis resolves over a variable period of time (days to months) after cessation of therapy. Therefore, a conservative approach to this condition is advocated, but a prolonged follow-up may be necessary. A personalized assessment of factors predisposing to ceftriaxone-associated biliary pseudolithiasis before prescribing the drug can allow to minimize the risk of developing it, with significant advantages in terms of human and economic costs.

A point mutation (P56S) in the gene-encoding vesicle-associated membrane-protein-associated protein B (VAPB) leads to an autosomal-dominant form of amyotrophic lateral sclerosis (ALS), classified as ALS-8. The mutant VAPB is characterized by ER-associated aggregates that lead to a complete reorganization of ER structures. Growing evidences suggest VAPB involvement in ALS pathomechanisms. In fact, numerous studies demonstrated VAPB alteration also in sporadic ALS (sALS) and showed the presence of its aggregates when others ALS-related gene are mutant. Recently, the identification of new biomarkers in peripheral blood mononuclear cells (PBMCs) has been proposed as a good noninvasive option for studying ALS. Here, we evaluated VAPB as a possible ALS pathologic marker analyzing PBMCs of sALS patients. Immunofluorescence analysis (IFA) showed a peculiar pattern of VAPB aggregates in sALS, not evident in healthy control (HC) subjects and in Parkinson&rsquo, s disease (PD) PBMCs. This specific pattern led us to suppose that VAPB could be misfolded in sALS. The data indirectly confirmed by flow cytometry assay (FCA) showed a reduction of VAPB fluorescent signals in sALS. However, our observations were not associated with the presence of a genetic mutation or altered gene expression of VAPB. Our study brings further evidences of the VAPB role in ALS as a diagnostic biomarker.

Non-alcoholic fatty liver disease (NAFLD) in children is becoming a major health concern. A "multiple-hit" pathogenetic model has been suggested to explain the progressive liver damage that occurs among children with NAFLD. In addition to the accumulation of fat in the liver, insulin resistance (IR) and oxidative stress due to genetic/epigenetic background, unfavorable lifestyles, gut microbiota and gut-liver axis dysfunction, and perturbations of trace element homeostasis have been shown to be critical for disease progression and the development of more severe inflammatory and fibrotic stages [non-alcoholic steatohepatitis (NASH)]. Simple clinical and laboratory parameters, such as age, history, anthropometrical data (BMI and waist circumference percentiles), blood pressure, surrogate clinical markers of IR (acanthosis nigricans), abdominal ultrasounds, and serum transaminases, lipids and glucose/insulin profiles, allow a clinician to identify children with obesity and obesity-related conditions, including NAFLD and cardiovascular and metabolic risks. A liver biopsy (the "imperfect" gold standard) is required for a definitive NAFLD/NASH diagnosis, particularly to exclude other treatable conditions or when advanced liver disease is expected on clinical and laboratory grounds and preferably prior to any controlled trial of pharmacological/surgical treatments. However, a biopsy clearly cannot represent a screening procedure. Advancements in diagnostic serum and imaging tools, especially for the non-invasive differentiation between NAFLD and NASH, have shown promising results, e.g., magnetic resonance elastography. Weight loss and physical activity should be the first option of intervention. Effective pharmacological treatments are still under development; however, drugs targeting IR, oxidative stress, proinflammatory pathways, dyslipidemia, gut microbiota and gut liver axis dysfunction are an option for patients who are unable to comply with the recommended lifestyle changes. When morbid obesity prevails, bariatric surgery should be considered.

This report summarizes the clinical features and the indications for treating HCV infection in immunocompromised and transplanted patients in the Direct Acting Antiviral drugs era.

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism. ispartof: Journal of Clinical Investigation vol:127 issue:3 pages:942-953 ispartof: location:United States status: published

Hepatitis E virus (HEV) was discovered in the 1980s and has been considered as being confined to developing countries. The purpose of this critical review was to determine the reported HEV seroprevalence rates in Italy, to identify predisposing factors and individuals at risk and to assess possible importation of HEV by immigrants. A critical review of 159 articles published in PubMed from 1994 to date was done. Only 27 original reports of 50 or more subjects, written in the English or Italian language, were included. Over three decades, the HEV seroprevalence varied from 0.12% to 49%, with the highest rates being reported from the central region of Italy. Risk factors included ingestion of raw pork or potentially contaminated food. The seroprevalence among immigrants ranged from 15.3% to 19.7% in Apulia. Italy has a population of 60 656 000; the total number of individuals surveyed was only 21.882 (0.036%). A national epidemiological survey program is needed to capture more comprehensive seroprevalence data. Keywords: Hepatitis E infection, Epidemiology, Seroprevalence, Risk factors, Immigrants, Italy

Diagnosis of de novo autoimmune hepatitis (AIH) after orthotopic liver transplantation (OLT) is challenging especially in the absence of hyper-γ-globulinemia. Circulating autoantibodies are not sensitive nor specific in de novo AIH but when positive increase the diagnostic probability. We report the discovery of novel liver microsomal (LM) autoantibodies against CYP-2C19 in a 9-year-old boy with “de novo” AIH developed 7 years after OLT. Graft dysfunction presented with hypertransaminasemia (up to 400 IU/L), while serum γ-globulins remained within the normal range for age. Liver histology and response to high dose prednisone (2 mg/kg/day) with the addition of azathioprine therapy further supported the diagnosis of de novo AIH. Autoantibodies investigation by indirect immunofluorescence (IF) on rodent tissues showed a novel staining pattern involving the pericentral liver zone and sparing the renal tissue. Human but not rat liver proteins immunoblotting allowed us to characterize the novel LM antibodies and to identify CYP-2C19 as human antigen. The finding offers insights into the controversial discussion about autoimmunity versus alloreactivity with regard to the pathogenesis of de novo AIH. Correct information on human versus rat tissue antigens tested by methods other than IF for antibodies detection may have significant implications for the correct diagnosis and management of patients followed up after OLT.

Recent studies have shown that infants with intrauterine growth restriction (IUGR) undergo catch-up growth during infancy. The aim of our study was to evaluate the postnatal growth in a cohort of IUGR infants born in a tertiary-level Obstetric University Hospital of Northern Sardinia. An observational retrospective study was conducted on 12 IUGR (group A) and 12 control infants (group B) by measuring the anthropometric parameters of weight (W), length (L) and head circumference (HC) from birth to the 3rd postnatal year. At birth, significant differences were found between group A and group B with regard to all the auxological parameters (W, mean 1846.6 versus 3170.8 g, p < 0.0001; HC, 30.1 versus 34.4 cm, p < 0.0001; L, mean 43.4 versus 49.4 cm, p < 0.0001). During the 1st year, 8 of 12 (70%) IUGR infants exhibited a significant catch-up growth in the 3 anthropometric parameters and a regular growth until the 3rd year of follow-up. The majority but not all infants born with IUGR in our series showed significant postnatal catch-up growth essentially during the first 12 months of life. An improved knowledge of the causes of IUGR will help to develop measures for its prevention and individualized treatment.

Background: The HLA DRB1*08 allele associated with primary biliary cholangitis (PBC) among Caucasians is of low frequency in the Sardinian population. Objective: The aim of our study was to type a cohort of PBC patients from the island of Sardinia for HLA class II antigens. Methods: Twenty Sardinian patients affected by PBC, 14 with autoimmune hepatitis (AIH) and 89 healthy controls (HCs) were typed for HLA class II alleles by dot-blot analysis. Results: The PBC-associated HLA DRB1*08 allele was detected in none of the studied individuals. The DRB1*0301–DQB1*0201 was the prevalent HLA haplotype, detected in 19 (47.5%) out of 40 PBC haplotypes (OR = 3.0; 95% CI 1.5–6.2) and in 11 (39.3%) out of 28 AIH haplotypes (OR = 2.2; 95% CI 0.94–5.0), but in only 41 (23%) out of 178 HC haplotypes. Moreover, PBC patients showed an increased frequency of homozygosity for the DQB1*0201 allele (35% compared with 6.7% of the HCs; OR = 7.5; 95% CI 2.2–25.7). The frequency of the DRB1*11 allele in the PBC group was about half of that seen in the Sardinian HCs (7.5% vs 15.7%) (p = ns). Conclusions: Our study confirmed the low frequency of the HLA DRB1*08 allele among Sardinians, either in the general population or PBC patients. The high prevalence of the HLA DRB1*0301–DQB1*0201 haplotype is a distinctive genetic feature of PBC among Sardinians. Our study strengthens the hypothesis that still unknown genetic, epigenetic, and environmental factors must be involved in the pathogenesis of different HLA-associated liver diseases, and it represents a pathfinder that warrants exploration in a future extensive study.

Biliary atresia is a severe and progressive inflammatory process of unknown cause, which initially involves the extrahepatic bile ducts but which quickly proceeds towards the intrahepatic bile tree leading rapidly to biliary cirrhosis. Biliary atresia is the major reason for liver transplantation during childhood. The extrahepatic bile ducts in biliary atresia become connective fibrotic cords which is irreversibly damaged.

Biliary atresia (BA) is notable for marked ductular reaction and rapid development of fibrosis. Activation of the Hedgehog (Hh) pathway promotes the expansion of populations of immature epithelial cells that coexpress mesenchymal markers and may be profibrogenic. We examined the hypothesis that in BA excessive Hh activation impedes ductular morphogenesis and enhances fibrogenesis by promoting accumulation of immature ductular cells with a mesenchymal phenotype. Livers and remnant extrahepatic ducts from BA patients were evaluated by quantitative reverse-transcription polymerase chain reaction (QRT-PCR) and immunostaining for Hh ligands, target genes, and markers of mesenchymal cells or ductular progenitors. Findings were compared to children with genetic cholestatic disease, age-matched deceased donor controls, and adult controls. Ductular cells isolated from adult rats with and without bile duct ligation were incubated with Hh ligand-enriched medium ± Hh-neutralizing antibody to determine direct effects of Hh ligands on epithelial to mesenchymal transition (EMT) marker expression. Livers from pediatric controls showed greater innate Hh activation than adult controls. In children with BA, both intra- and extrahepatic ductular cells demonstrated striking up-regulation of Hh ligand production and increased expression of Hh target genes. Excessive accumulation of Hh-producing cells and Hh-responsive cells also occurred in other infantile cholestatic diseases. Further analysis of the BA samples demonstrated that immature ductular cells with a mesenchymal phenotype were Hh-responsive. Treating immature ductular cells with Hh ligand-enriched medium induced mesenchymal genes; neutralizing Hh ligands inhibited this. Conclusion: BA is characterized by excessive Hh pathway activity, which stimulates biliary EMT and may contribute to biliary dysmorphogenesis. Other cholestatic diseases show similar activation, suggesting that this is a common response to cholestatic injury in infancy. (HEPATOLOGY 2011;)

In short small-for-gestational-age (SGA) patients, the exon 3-deleted(d3)/full-length (fl)-GHR polymorphism was associated with responsiveness to GH therapy (30-48 microg/kg.d); however, these results were not confirmed for higher GH doses (56-66 microg/kg.d). We hypothesized that higher doses would mask the lower dose differences.Our objective was to evaluate, in short SGA patients, 2-yr growth response to GH therapy (32.1 +/- 3.8 microg/kg.d) according to exon d3/fl-GHR genotypes.This was a 2-yr follow-up study.There was a total of 60 short SGA children (d3/d3 n = 8, d3/fl n = 23, and fl/fl n = 29). There were 11 children that entered puberty during the second follow-up year. Results were evaluated for all patients (group A1, n = 60, 7.7 +/- 2.7 yr) and for patients who remained prepubertal (group A2, n = 49, 6.9 +/- 2.2 yr).Patients were followed by a single clinical team, and exon d3/fl-GHR genotypes were determined and analyzed in the same hospital.In groups A1 and A2, growth velocity significantly (P0.0001) increased during the first and second years of therapy, as did height sd score (SDS). These increases were similar in each exon d3/fl-GHR genotype. Total 2-yr height gain (cm, SDS) did not differ statistically among genotypes: group A1, 15.0 +/- 2.0 cm and 1.15 +/- 0.45 SDS in d3/d3, 16.0 +/- 2.4 cm and 1.17 +/- 0.51 SDS in d3/fl, 16.1 +/- 2.4 cm and 1.15 +/- 0.53 SDS in fl/fl; and group A2, 15.4 +/- 2.0 cm and 1.03 +/- 0.42 SDS in d3/d3, 15.6 +/- 2.1 cm and 1.22 +/- 0.51 in d3/fl, and 16.2 +/- 2.6 cm and 1.21 +/- 0.56 SDS in fl/fl.These results did not confirm our hypothesis and show that, in short SGA children, 2-yr growth response to GH therapy 32.1 +/- 3.8 microg/kg.d was similar for each exon d3/fl-GHR genotype carried, as occurred in our previous study using 66 microkg.d.

Biliary atresia (BA) is an infantile inflammatory cholangiopathy of unknown etiology although epidemiologic studies and animal models utilizing rotavirus (RV) have suggested a role for viral infection. Proinflammatory and profibrotic cytokines have been detected in infants with BA. The purpose of our study was to investigate the susceptibility of human cholangiocytes (H69 cells) to infection with RRV and to determine if this infection resulted in cytokine secretion. Infection of H69 cells by RRV was noncytolytic and resulted in a time-dependent increase in the release of both infectious virions and cytokines IL-6 and IL-8 into the supernate. The greatest difference in cytokine supernatant levels between infected and mock-infected cells was noted at 24 hours postinfection (h p.i.) for IL-8, 556 ± 111 versus 77 ± 68 pg/mL (p<0.0001), and at 48 h p.i. for IL-6, 459 ± 64 versus 67 ± 2 pg/mL (p<0.0001). Production of both cytokines following RRV infection was significantly reduced by pretreating the H69 cells with inhibitors of mitogen-activated protein kinase (MAPK).Conclusion. RRV can infect human cholangiocytes resulting in the production of proinflammatory and profibrotic cytokines via the MAPK pathway. RRV-infected H69 cells could be a useful model system for investigating the viral hypothesis of BA.

The d3/fl-GH receptor (d3/fl-GHR, exon 3-deleted/full-length GHR) has recently been associated with responsiveness to GH therapy.The objective of the study was to evaluate whether the d3/fl-GHR genotypes influence the intensity of spontaneous and/or GH therapy-stimulated growth in small-for-gestational-age (SGA) patients.This was a 2-yr prospective, controlled, randomized trial.Thirty Spanish hospitals participated. Auxologic and GH secretion evaluation was hospital based, whereas molecular analyses and auxologic data computation were centralized.Patients included 170 short SGA children: 140 remained prepubertal and 30 entered puberty during the second follow-up year.Eighty-six were treated with GH (66 microg/kg.d) for 2 yr and 84 were not treated.Previous and 2-yr follow-up auxologic data were recorded at each hospital, d3/fl-GHR genotypes determined, and data analyzed for patients who remained prepubertal (group 1, 68 GH treated and 72 non-GH treated) and for all the patients (group 2).In group 1 GH-treated patients, growth velocity, and height-sd score during the first and second years, total 2-yr height gain (18.5 +/- 2.4 cm in d3/d3; 18.4 +/- 2.6 in d3/fl; 19.5 +/- 2.3 in fl/fl), Delta 2-yr height increase (9.1 +/- 2.4 cm in d3/d3; 9.4 +/- 3.0 in d3/fl; 10.4 +/- 2.1 in fl/fl), first-year growth prediction and studentized residual values (0.08 +/- 1.26 in d3/d3; 0.28 +/- 1.21 in d3/fl; 0.67 +/- 0.95 in fl/fl) did not differ among the d3/fl-GHR genotypes. In group 1 non-GH-treated patients, neither growth velocity nor height-sd score changed significantly, and values were similar in each d3/fl-GHR genotype. Results in all patients (group 2) were similar to those in group 1.In short non-GH-deficient SGA children, both spontaneous growth rate and responsiveness to 66 microg/k.d GH therapy were similar for each d3/fl-GHR genotype carried.

The native structure and distribution of gliadin epitopes responsible for Celiac Sprue (CS) may be influenced by cereal food processing. This work was aimed at showing the capacity of probiotic VSL#3 to decrease the toxicity of wheat flour during long-time fermentation. VSL#3 (109 cfu/ml) hydrolyzed completely the α2-gliadin-derived epitopes 62–75 and 33-mer (750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and mass spectrometry analyses showed an almost complete degradation of gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins non-hydrolyzed during fermentation by VSL#3 were subjected to peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry). Search for several epitopes showed the only presence of α2-gliadin-fragment 62–75 at a very low concentration (sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins extracted from the chemically acidified dough (control), VSL#3 pre-digested gliadins caused a less pronounced reorganization of the intracellular F-actin which was mirrored by an attenuated effect on intestinal mucosa permeability. The release of zonulin from intestinal epithelial cells treated with gliadins was considerably lower when digested with VSL#3. Agglutination test on K 562 (S) cells showed that the PT-digest of wheat flour treated with VSL#3 increased the Minimal Agglutinating Activity of ca. 100 times. Wheat proteins were extracted from doughs and subjected to PT digestion. Compared to PT-digest from chemically acidified dough, celiac jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3 did not show an increase of the infiltration of CD3+ intraepithelial lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance during food processing to produce pre-digested and tolerated gliadins for increasing the palatability of gluten-free products.

BACKGROUND—The cytoskeleton actin network of intestinal microvilli has been found to be rapidly impaired after gluten challenge in coeliac disease (CD). The aim of this study was to investigate the presence of an immune reaction towards cytoskeleton structures such as actin filaments in CD. METHODS—Eighty three antiendomysial antibody positive CD patients (52 children and 31 adults) were studied at our outpatient clinics from 1996 to 1998 using indirect immunofluorescence, ELISA, and western blotting for antiactin (AAA) and antitissue transglutaminase (TGA) antibodies before and after a gluten free diet (GFD). Sixteen patients with smooth muscle antibody positive autoimmune hepatitis, 21 with inflammatory bowel diseases, seven with small bowel bacterial overgrowth, and 60 healthy subjects were studied as controls. RESULTS—Fifty nine of 83 CD patients (28/31 adults (90.3%); 31/52 children (59.6%)) were positive for IgA and/or IgG AAA. Seventy seven (92.7%) were positive for IgA TGA. IgA AAA were strongly correlated with more severe degrees of intestinal villous atrophy (p

OBJECTIVE—To report the long-term follow-up of three nonpancreatectomized patients with persistent hyperinsulinemic hypoglycemia of infancy due to mutations in the ABCC8 gene. RESEARCH DESIGN AND METHODS—Oral glucose tolerance test (OGTT) and venous 24-h glucose-insulin profile were performed yearly from adolescence. RESULTS—Patient 1 (now aged 31 years) developed insulin-dependent diabetes at the age of 25 years. In patient 2 (now aged 17 years), impaired fasting glucose and a diabetic OGTT response with normal A1C values have been observed since the age of 10 years. In patient 3 (now aged 24 years), intolerant OGTT response and hyperglycemic episodes with normal A1C have been observed since the age of 16 years. All patients presented relatively low insulin levels during hyperglycemia, normal BMI, and negative autoantibodies (GAD antibody, insulinoma-associated protein 2, and islet cell antibody). CONCLUSIONS—Development of glucose metabolism impairment ranging from glucose intolerance to insulin-dependent diabetes is observed in the evolution of these patients.

Background & Aims: Liver disease has been described in 10%–15% of patients with autoimmune polyglandular syndrome type 1 (APS-1). After the discovery of cytochrome P450 1A2 (CYP1A2) as a hepatocellular autoantigen in liver-kidney microsomal autoantibody (LKM)-positive patients with APS-1, the investigation of antiliver antibodies was extended to 11 Sardinian patients with APS-1. Methods: Indirect immunofluorescence and Western blotting analysis were performed to study the antiliver antibodies. Results: Immunofluorescence revealed LKM antibodies in 3 patients with APS-1, 1 of whom died of fulminant hepatitis. Western blotting showed a liver microsomal protein band of approximately 51 kilodaltons in the LKM-positive sera of these 3 patients. Western blotting performed with recombinant cytochrome P450 enzymes allowed the identification of CYP2A6 as a specific target antigen. Conclusions: LKM antibodies in APS-1 sera are specifically directed against CYP1A2 or CYP2A6, but their diagnostic and prognostic significance for liver disease remain to be determined. GASTROENTEROLOGY 1998;114:324-328

Autoantibodies directed against proteins of the adrenal cortex and the liver were studied in 88 subjects of Sardinian descent, namely six patients with autoimmune polyendocrine syndrome type 1 (APS1), 22 relatives of APS1 patients, 40 controls with other autoimmune diseases, and 20 healthy controls. Indirect immunofluorescence, using tissue sections of the adrenal cortex, revealed a cytoplasmatic staining pattern in 4 of 6 patients with APS1. Western blotting with adrenal mitochondria identified autoantigens of 54 kDa and 57 kDa, Western blotting with placental mitochondria revealed a 54-kDa autoantigen. The 54-kDa protein was recognized by 4 of 6 patients with APS1 both in placental and adrenal tissue, whereas the 57-kDa protein was detected only by one serum. Using recombinant preparations of cytochrome P450 proteins, the autoantigens were identified as P450 scc and P450 c17. One of six APS1 patients suffered from chronic hepatitis. In this patient, immunofluorescence revealed a centrolobular liver and ...

This study reports the molecular characterisation of the bilirubin UDP-glucuronosyl-transferase gene (UGT1) in a group of patients of Sardinian descent with Crigler-Najjar syndrome type I and their relatives. Sequence analysis of both UGT1A exon 1 and common exons 2-5 was performed in all patients, leading to the detection of AF170 and a novel mutation (470insT), both residing in UGT1A exon 1. All but two heterozygotes for the AF170 mutation showed normal serum bilirubin levels. These two subjects were also heterozygous for the sequence variation A(TA)7TAA in the promoter region of the UGT1A gene.

A polymorphism in the human GH receptor gene (d3/fl-GHR) resulting in genomic deletion of exon 3 has been associated with the degree of height increase in response to GH therapy.The objective of the study was to evaluate the frequencies of d3/fl-GHR polymorphism genotypes in control and short small-for-gestational-age (SGA) populations.An adult control population with heights normally distributed (ACPNH) between -2 and +2 sd score (SDS) and a short non-GH-deficient SGA child population were selected.Thirty Spanish hospitals participated in the selection of the short non-GH-deficient SGA children in the setting of a controlled, randomized trial, and one of these hospitals selected the ACPNH. CONTROLS AND PATIENTS: Two hundred eighty-nine adult subjects of both sexes constituted the ACPNH and 247 children and adolescents of both sexes the short SGA patients.Heights and weights were recorded in the ACPNH, and auxologic and biochemical data were recorded at each hospital for the SGA patients; d3/fl-GHR genotypes were determined and data analyzed in a single hospital.In short SGA patients, d3/fl-GHR genotype frequencies were significantly different from those in ACPNH, with a higher frequency of fl/fl genotype (P0.0001). In ACPNH, a trend toward diminished d3/d3 genotype frequency was observed in the shortest height group (heightor=-1 SDS andor=-2 SDS, n = 60).Our data showed significant differences in the frequency distribution of the d3/fl-GHR genotypes between a normally distributed adult height population and short SGA children, with the biologically less active fl/fl genotype being almost twice as frequent in SGA patients. These data suggest that the d3/fl-GHR polymorphism might be considered among the factors that contribute to the phenotypic expression of growth.

Zonulin, a protein that modulates intestinal permeability, is upregulated in several autoimmune diseases and is involved in the pathogenesis of autoimmune diabetes in the BB/Wor animal model of the disease. To verify the association between serum zonulin levels and in vivo intestinal permeability in patients with type 1 diabetes, both parameters were investigated in different stages of the autoimmune process. Forty-two percent (141 of 339) of the patients had abnormal serum zonulin levels, as compared with age-matched control subjects. The increased zonulin levels correlated with increased intestinal permeability in vivo and changes in claudin-1, claudin-2, and myosin IXB genes expression, while no changes were detected in ZO1 and occludin genes expression. When tested in serum samples collected during the pre‐type 1 diabetes phase, elevated serum zonulin was detected in 70% of subjects and preceded by 3.5 0.9 years the onset of the disease in those patients who went on to develop type 1 diabetes. Combined, these results suggest that zonulin upregulation is associated with increased intestinal permeability in a subgroup of type 1 diabetic patients. Zonulin upregulation seems to precede the onset of the disease, providing a possible link between increased intestinal permeability, environmental exposure to non‐self antigens, and the development of autoimmunity in genetically susceptible individuals. Diabetes 55:1443‐1449, 2006

Objective. Little is known about the interaction of gliadin with intestinal epithelial cells and the mechanism(s) through which gliadin crosses the intestinal epithelial barrier. We investigated whether gliadin has any immediate effect on zonulin release and signaling. Material and methods. Both ex vivo human small intestines and intestinal cell monolayers were exposed to gliadin, and zonulin release and changes in paracellular permeability were monitored in the presence and absence of zonulin antagonism. Zonulin binding, cytoskeletal rearrangement, and zonula occludens-1 (ZO-1) redistribution were evaluated by immunofluorescence microscopy. Tight junction occludin and ZO-1 gene expression was evaluated by realtime polymerase chain reaction (PCR). Results. When exposed to gliadin, zonulin receptor-positive IEC6 and Caco2 cells released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell cytoskeleton, loss of occludin-ZO1 protein � /protein interaction, and increased monolayer permeability. Pretreatment with the zonulin antagonist FZI/0 blocked these changes without affecting zonulin release. When exposed to luminal gliadin, intestinal biopsies from celiac patients in remission expressed a sustained luminal zonulin release and increase in intestinal permeability that was blocked by FZI/0 pretreatment. Conversely, biopsies from non-celiac patients demonstrated a limited, transient zonulin release which was paralleled by an increase in intestinal permeability that never reached the level of permeability seen in celiac disease (CD) tissues. Chronic gliadin exposure caused down-regulation of both ZO-1 and occludin gene expression. Conclusions. Based on our results, we concluded that gliadin activates zonulin signaling irrespective of the genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules.

Background and aims: Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability. Methods: The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers. Results: Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release. Conclusions: Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.

Objective Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.