Wheat DOF transcription factors TaSAD and WPBF regulate glutenin gene expression in cooperation with SPA.

Grain storage proteins (GSPs) quantity and composition determine the end-use value of wheat flour. GSPs consists of low-molecular-weight glutenins (LMW-GS), high-molecular-weight glutenins (HMW-GS) and gliadins. GSP gene expression is controlled by a complex network of DNA-protein and protein-protein interactions, which coordinate the tissue-specific protein expression during grain development. The regulatory network has been most extensively studied in barley, particularly the two transcription factors (TFs) of the DNA binding with One Finger (DOF) family, barley Prolamin-box Binding Factor (BPBF) and Scutellum and Aleurone-expressed DOF (SAD). They activate hordein synthesis by binding to the Prolamin box, a motif in the hordein promoter. The BPBF ortholog previously identified in wheat, WPBF, has a transcriptional activity in expression of some GSP genes. Here, the wheat ortholog of SAD, named TaSAD, was identified. The binding of TaSAD to GSP gene promoter sequences in vitro and its transcriptional activity in vivo were investigated. In electrophoretic mobility shift assays, recombinant TaSAD and WPBF proteins bound to cis-motifs like those located on HMW-GS and LMW-GS gene promoters known to bind DOF TFs. We showed by transient expression assays in wheat endosperms that TaSAD and WPBF activate GSP gene expression. Moreover, co-bombardment of Storage Protein Activator (SPA) with WPBF or TaSAD had an additive effect on the expression of GSP genes, possibly through conserved cooperative protein-protein interactions.

Critical structural elements for the antigenicity of wheat allergen LTP1 (Tri a 14) revealed by site-directed mutagenesis.

Lipid transfer proteins (LTPs) were identified as allergens in a large variety of pollens and foods, including cereals. LTPs belong to the prolamin superfamily and display an α-helical fold, with a bundle of four α-helices held together by four disulfide bonds. Wheat LTP1 is involved in allergic reactions to food. To identify critical structural elements of antibody binding to wheat LTP1, we used site-directed mutagenesis on wheat recombinant LTP1 to target: (i) sequence conservation and/or structure flexibility or (ii) each disulfide bond. We evaluated the modifications induced by these mutations on LTP1 secondary structure by synchrotron radiation circular dichroism and on its antigenicity with patient's sera and with mouse monoclonal antibodies. Disruption of the C28-C73 disulfide bond significantly affected IgE-binding and caused protein denaturation, while removing C13-C27 bond decreased LTP1 antigenicity and slightly modified LTP1 overall folding. In addition, we showed Lys72 to be a key residue; the K72A mutation did not affect global folding but modified the local 3D structure of LTP1 and strongly reduced IgE-binding. This work revealed a cluster of residues (C13, C27, C28, C73 and K72), four of which embedded in disulfide bonds, which play a critical role in LTP1 antigenicity.

Colon capsule endoscopy in clinical practice: lessons from a national 5-year observational prospective cohort.

Background and study aims Colon capsule endoscopy (CCE) has been proposed as an alternative to colonoscopy for screening patients at average risk of colorectal cancer (CRC). A prospective national cohort was developed to assess relevance of CCE in real-life practice and its short- and long-term impacts on clinical management. Patients and methods All patients who underwent a CCE in France were prospectively enrolled from January 2011 to May 2016 and reached annually by phone until May 2017. All CCE and colonoscopy reports were systematically collected. Results During the study period, 689 CCEs were analyzed from 14 medical centers. Median follow-up time was 35 months [IQR: 12-50]. Indication for CCE was mainly for elderly patients (median age: 70 years, IQR: [61-79]) due to anesthetic or colonoscopy contraindication (n = 307; 44.6 %). Only 337 CCEs (48.9 %) were both complete and with adequate bowel preparation. Advanced neoplasia (adenoma with high-grade dysplasia or CRC) was diagnosed following 32 CCEs (4.6 %). Among patients who underwent colonoscopy or therapeutic surgery following CCE, 18.8 % of all advanced neoplasias (6/32) had not been diagnosed by CCE mainly due to technical issues. Performing a colonoscopy in the case of significant polyps or insufficient bowel cleansing or after an incomplete CCE allowed the diagnosis of 96.9 % of all identified advanced neoplasias (31/32). Conclusions Outside the scope of academic trials, improvement is needed to increase the reliability of CCE as less than half were considered optimal i. e. complete with adequate bowel cleansing. Most of missed colonic advanced neoplasia were due to incomplete CCE with distal neoplasia location.

The bZIP transcription factor SPA Heterodimerizing Protein represses glutenin synthesis in Triticum aestivum.

The quality of wheat grain is mainly determined by the quantity and composition of its grain storage proteins (GSPs). Grain storage proteins consist of low- and high-molecular-weight glutenins (LMW-GS and HMW-GS, respectively) and gliadins. The synthesis of these proteins is essentially regulated at the transcriptional level and by the availability of nitrogen and sulfur. The regulation network has been extensively studied in barley where BLZ1 and BLZ2, members of the basic leucine zipper (bZIP) family, activate the synthesis of hordeins. To date, in wheat, only the ortholog of BLZ2, Storage Protein Activator (SPA), has been identified as playing a major role in the regulation of GSP synthesis. Here, the ortholog of BLZ1, named SPA Heterodimerizing Protein (SHP), was identified and its involvement in the transcriptional regulation of the genes coding for GSPs was analyzed. In gel mobility shift assays, SHP binds cis-motifs known to bind to bZIP family transcription factors in HMW-GS and LMW-GS promoters. Moreover, we showed by transient expression assays in wheat endosperm that SHP acts as a repressor of the activity of these gene promoters. This result was confirmed in transgenic lines overexpressing SHP, which were grown with low and high nitrogen supply. The phenotype of SHP-overexpressing lines showed a lower quantity of both LMW-GS and HMW-GS, while the quantity of gliadin was unchanged, whatever the nitrogen availability. Thus, the gliadin/glutenin ratio was increased, which suggests that gliadin and glutenin genes may be differently regulated.

Thoracic skeletal anomalies following surgical treatment of esophageal atresia. Lessons from a national cohort.

INTRODUCTION: Thoracotomy as surgical approach for esophageal atresia treatment entails the risk of deformation of the rib cage and consequently secondary thoracogenic scoliosis. The aim of our study was to assess these thoracic wall anomalies on a large national cohort and search for factors influencing this morbidity. MATERIALS AND METHODS: Pediatric surgery departments from our national network were asked to send recent thoracic X-ray and operative reports for patients born between 2008 and 2010 with esophageal atresia. The X-rays were read in a double-blind manner to detect costal and vertebral anomalies. RESULTS: Among 322 inclusions from 32 centers, 110 (34.2%) X-rays were normal and 25 (7.7%) displayed thoracic malformations, including 14 hemivertebrae. We found 187 (58.1%) sequelae of surgery, including 85 costal hypoplasia, 47 other types of costal anomalies, 46 intercostal space anomalies, 21 costal fusions and 12 scoliosis, with some patients suffering from several lesions. The rate of patients with these sequelae was not influenced by age at intervention, weight at birth, type of atresia, number of thoracotomy or size of the center. The rate of sequelae was higher following a classical thoracotomy (59.1%), whatever the way that thoracotomy was performed, compared to nonconverted thoracoscopy (22.2%; p=0.04). CONCLUSION: About 60 % of the patients suffered from a thoracic wall morbidity caused by the thoracotomy performed as part of surgical treatment of esophageal atresia. Minimally invasive techniques reduced thoracic wall morbidity. Further studies should be carried out to assess the potential benefit of minimally invasive approaches to patient pulmonary functions and on the occurrence of thoracogenic scoliosis in adulthood. LEVELS OF EVIDENCE: Level III retrospective comparative treatment study.

A chimeric IgE that mimics IgE from patients allergic to acid-hydrolyzed wheat proteins is a novel tool for in vitro allergenicity assessment of functionalized glutens.

BACKGROUND: Acid-hydrolyzed wheat proteins (acid-HWPs) have been shown to provoke severe allergic reactions in Europe and Japan that are distinct from classical wheat allergies. Acid-HWPs were shown to contain neo-epitopes induced by the deamidation of gluten proteins. However, products with variable rates of deamidation can be found. OBJECTIVES: In this work, we studied the effect of the extent of wheat proteins deamidation on its allergenicity. A recombinant chimeric IgE was produced and compared to patients' IgE for its capacity to assess the IgE-mediated triggering potential of acid-HWPs. METHODS: Sera from acid-HWP allergic patients were analyzed via ELISA and a functional basophil assay for their IgE reactivity to wheat proteins with different deamidation levels. A chimeric mouse/human IgE (chIgE-DG1) specific for the main neo-epitope, QPEEPFPE, involved in allergy to acid-HWPs was characterized with respect to its functionality and its reactivity compared to that of patients' IgE. RESULTS: Acid-HWPs with medium (30%) and high (50-60%) deamidation levels displayed a markedly stronger IgE binding and capacity to activate basophils than those of samples with weak (15%) deamidation levels. The monoclonal chIgE-DG1 allowed basophil degranulation in the presence of deamidated wheat proteins. ChIgE-DG1 was found to mimic patients' IgE reactivity and displayed the same ability to rank acid-HWP products in a degranulation assay. CONCLUSION: Increasing the deamidation level of products from 15% to 60% resulted in an approximately 2-fold increase in their antigenicity and a 100-fold increase in their eliciting potential. The chimeric ChIgE-DG1 may be a useful tool to evaluate functionalized glutens for their allergenic potential. By mimicking patient sera reactivity, chIgE-DG1 also provided data on the patients' IgE repertoire and on the functionality of certain repeated epitopes in gluten proteins.

Prevalence of Barrett Esophagus in Adolescents and Young Adults With Esophageal Atresia.

OBJECTIVE: To study the prevalence of Barrett esophagus (BE) (gastric and/or intestinal metaplasia) in adolescents treated for esophageal atresia (EA). SUMMARY OF BACKGROUND DATA: EA patients are at high risk of BE. METHODS: This multicenter prospective study included EA patients aged 15 to 19 years. All eligible patients were proposed an upper endoscopy with multistaged esophageal biopsies under general anesthesia. Histological suspicion of metaplasia was confirmed centrally. RESULTS: One hundred twenty patients [mean age, 16.5 years (±1.4)] were included; 70% had been treated for gastroesophageal reflux disease (GERD) during infancy. At evaluation, 8% were undernourished, 41% had received antireflux surgery, and 41% presented with GERD symptoms, although only 28% were receiving medical treatment. Esophagitis was found at endoscopy in 34% and confirmed at histology in 67%. BE was suspected after endoscopy in 37% and was confirmed by histology for 43% of patients (50 gastric and 1 intestinal metaplasia). No endoscopic or histological anomalies were found at the anastomosis site. BE was not significantly related to clinical symptoms. In multivariate analysis, BE was associated with EA without fistula (P = 0.03), previous multiple antireflux surgery (P = 0.04), esophageal dilation (P = 0.04), suspicion of BE at endoscopy (P < 0.001), and histological esophagitis (P = 0.02). CONCLUSIONS: Patients with EA are at high risk of persistent GERD and BE. The development of BE is related to GERD history. Long-term systematic follow-up of the esophageal mucosa including multistaged biopsies is required, even in asymptomatic patients. (NCT02495051).

Structural Basis of IgE Binding to α- and γ-Gliadins: Contribution of Disulfide Bonds and Repetitive and Nonrepetitive Domains.

Wheat products cause IgE-mediated allergies. The present study aimed to decipher the molecular basis of α- and γ-gliadin allergenicity. Gliadins and their domains, the repetitive N-terminal and the nonrepetitive C-terminal domains, were cloned and expressed in Escherichia coli. Their secondary structures and their IgE binding capacity were compared with those of natural proteins before and after reduction/alkylation. Allergenicity was evaluated with sera from patients who had a wheat food allergy or baker's asthma. The secondary structures of natural and recombinant proteins were slightly different. Compared with natural gliadins, recombinant proteins retained IgE binding but with reduced reactivity. Reduction/alkylation decreased IgE binding for both natural and recombinant gliadins. Although more continuous epitopes were identified in the N-terminal domains of α- and γ-gliadins, both the N-terminal and C-terminal domains contributed to IgE binding. As for other members of the prolamin superfamily, disulfide bonds appear to be of high importance for IgE binding.

Results from the French National Esophageal Atresia register: one-year outcome.

BACKGROUND: The aim of the present national prospective population-based study was to assess the early morbidity of esophageal atresia (EA). METHODS: All 38 multidisciplinary French centers that care for patients with EA returned a specific questionnaire about the 1-year outcome for each patient. This information was centralized, checked, and entered into a database. RESULTS: From the total population of 307 EA patients born in 2008 and 2009, data about the 1-year outcome were obtained from 301 (98%) patients, of whom 4% were lost to follow-up and 5% died. Medical complications occurred in 34% of the patients: anastomotic leaks (8%), recurrent tracheoesophageal fistula (4%), and anastomotic stenosis (22%); all of the latter group needed dilation (median, 2 dilations/patient). A new hospitalization was required for 59% of patients (2.5 hospitalizations/patient) for digestive (52%) or respiratory (48%) reasons. Twelve percent of patients required antireflux surgery at a median age of 164 days (range, 33-398 days), and 1% underwent an aortopexy for severe tracheomalacia. The weight/age Z-score was -0.8 (range, -5.5 to 3.7 months) at 12 months. Fifteen percent of patients were undernourished at 12 months of age, whereas 37% presented with respiratory symptoms and 15% had dysphagia at the last follow-up. Significant independent factors associated with medical complications were anastomotic esophageal tension (p = .0009) and presence of a gastrostomy (p = .0002); exclusive oral feeding at discharge was associated with a decreased risk of complications (p = .007). CONCLUSIONS: Digestive and respiratory morbidities remain frequent during the first year of life and are associated with difficult anastomosis and lack of full oral feeding.

Normalization of test and evaluation of biothreat detection systems: overcoming microbial air content fluctuations by using a standardized reagent bacterial mixture.

Test and evaluation of engineered biothreat agent detection systems ("biodetectors") are a challenging task for government agencies and industries involved in biosecurity and biodefense programs. In addition to user friendly features, biodetectors need to perform both highly sensitive and specific detection, and must not produce excessive false alerts. In fact, the atmosphere displays a number of variables such as airborne bacterial content that can interfere with the detection process, thus impeding comparative tests when carried out at different times or places. To overcome these bacterial air content fluctuations, a standardized reagent bacterial mixture (SRBM), consisting in a collection of selected cultivable environmental species that are prevalent in temperate climate bioaerosols, was designed to generate a stable, reproducible, and easy to use surrogate of bioaerosol sample. The rationale, design, and production process are reported. The results showed that 8.59; CI 95%: 8.46-8.72 log cfu distributed into vials underwent a 0.95; CI 95%: 0.65-1.26 log viability decay after dehydration and subsequent reconstitution, thus advantageously mimicking a natural bioaerosol sample which is typically composed of cultivable and uncultivable particles. Dehydrated SRBM was stable for more than 12months at 4°C and allowed the reconstitution of a dead/live cells aqueous suspension that is stable for 96h at +4°C, according to plate counts. Specific detection of a simulating biothreat agent (e.g. Bacillus atrophaeus) by immuno-magnetic or PCR assays did not display any significant loss of sensitivity, false negative or positive results in the presence of SRBM. This work provides guidance on testing and evaluating detection devices, and may contribute to the establishment of suitable standards and normalized procedures.

MS analysis and molecular characterization of Botrytis cinerea protease Prot-2. Use in bioactive peptides production.

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.

Molecular and immunological characterization of wheat Serpin (Tri a 33).

SCOPE: Several wheat proteins are responsible for food and respiratory allergies. Due to their large polymorphism, the allergenic potential of a number of them has not yet been precisely established. The aim of this work was to perform a thorough assessment of serpin (Tri a 33) allergenicity. METHODS AND RESULTS: Recombinant wheat Serpin-Z2B isoform (rSerpin-Z2B) was expressed in Escherichia coli. Synchrotron radiation circular dichroism data indicated that the recombinant serpin contains slightly more ß-strands than α-helix structures. IgE reactivity of sera from 103 patients with food allergy and 29 patients with Baker's asthma was evaluated using ELISA, a model of basophil activation and linear epitope mapping (Pepscan). Twenty percent of patients with food allergy to wheat and 31% of those with Baker's asthma displayed rSerpin-Z2B-specific IgE in ELISA. The protein was able to induce IgE-dependent basophil degranulation. The Pepscan experiment identified four regions involved in IgE binding to serpin. Heating the protein induced its irreversible denaturation and impaired IgE binding, revealing the predominance of conformational epitopes. CONCLUSION: This study confirms wheat serpin allergenicity and shows that recombinant serpin may be a marker of a broad spectrum of sensitization to wheat proteins.

Outcome of alimentary tract duplications operated on by minimally invasive surgery: a retrospective multicenter study by the GECI (Groupe d'Etude en Coeliochirurgie Infantile).

BACKGROUND: Alimentary tract duplications (ATD) are a rare cause of intestinal obstruction in childhood. There are many case reports but few series about laparoscopy or thoracoscopy for ATD. The aim of our study was to report the outcome of minimally invasive surgery (MIS) for ATD. METHODS: This was a retrospective multicenter study from the GECI (Groupe d'Etude en Coeliochirurgie Infantile). We reviewed the charts of 114 patients operated on by MIS for ATD from 1994 to 2009. RESULTS: Sixty-two patients (54 %) had a prenatal diagnosis. Forty-nine patients (43 %) were symptomatic before surgery: 33 of those patients (63 %) with postnatal diagnosis compared to 16 (25 %) with prenatal diagnosis (P < 0.01). In this last group, the median age at onset of symptoms was 16 days (range = 0-972). One hundred and two patients had laparoscopy (esophageal to rectal duplications) and 12 patients had thoracoscopy for esophageal duplications. The mean operative time was 90 min (range = 82-98). There were 32 (28 %) resection anastomoses, 55 (48 %) enucleations, and 27 (24 %) unroofings. The conversion rate was 32 %, and in a multivariate analysis, it was significantly higher, up to 41 % for patients weighing <10 kg (P < 0.01). Ten patients (8 %) had unintentional perioperative opening of the digestive tract during the dissection. Eight patients had nine postoperative complications, including six small bowel obstructions. The median length of hospital stay was 4 days (range = 1-21) without conversion and 6 days (range = 1-27) with conversion (P = 0.01). The median follow-up was 3 months (range = 1-120). Eighteen of the 27 patients who underwent partial surgery had an ultrasound examination during follow-up. Five (18 %) of them had macroscopic residue. CONCLUSION: This study showed that MIS for ATD is feasible with a low rate of complications. Patients with prenatal diagnosis should have prompt surgery to prevent symptoms, despite a high rate of conversion in small infants.

Wheat gliadins modified by deamidation are more efficient than native gliadins in inducing a Th2 response in Balb/c mice experimentally sensitized to wheat allergens.

SCOPE: Wheat gluten proteins such as gliadins constitute major food allergens. Gluten can be modified industrially by deamidation which increases its solubility and enhances its use as a food ingredient. Sensitization to deamidated gluten has been reported to cause severe allergic reactions with anaphylaxis. The aim of this study was therefore to compare the sensitization and elicitation potentials of native (NG) and deamidated (DG) gliadins. The reactivity pattern of mice IgE was also compared with that of DG-allergic patients. METHODS AND RESULTS: The ability of DG to sensitize Balb/c mice using intra-peritoneal administration with aluminium hydroxide as an adjuvant, and to elicit an allergic response after a challenge, was tested in comparison with NG. Mice sensitized with DG secreted higher levels of total IgE, IL-4, gliadin-specific IgE and IgG1 than mice sensitized with NG. By contrast, mice sensitized with NG produced higher levels of gliadin-specific IgG2a and INFγ. After a challenge, histamine levels were higher in mice sensitised with DG. CONCLUSIONS: DG can sensitize mice much more efficiently than NG. Moreover, this mouse model of allergy to DG revealed an IgE reactivity pattern against purified gliadins which was very similar to that of DG-allergic patients.

Prospective, randomized comparison of two small-bowel capsule endoscopy systems in patients with obscure GI bleeding.

BACKGROUND: Video capsule endoscopy is the first-intention examination in patients with obscure GI bleeding. The new MiroCam capsule, when using electric-field propagation for transmission, has been poorly evaluated in a clinical setting, in contrast with the PillCam SB2 capsule. OBJECTIVE: To evaluate the diagnostic concordance (κ value) between PillCam SB2 and MiroCam capsule examinations performed in the same patients. DESIGN AND SETTING: Prospective, randomized study in 7 endoscopy units. PATIENTS AND INTERVENTION: Eighty-three consecutive patients, ingesting the 2 capsules at a 1-hour interval. RESULTS: Seventy-three patients were analyzed (10 technical issues). There were 31 concordant negative cases (42.4%) and 30 concordant positive cases (41.1%). The study showed satisfactory diagnostic concordance between the 2 systems (κ = 0.66). In 12 patients (16.4%), the final diagnosis was different: 9 patients had positive findings on MiroCam examination but no image detected with PillCam SB2, 2 had positive findings on PillCam examination only, and 1 patient had 2 different diagnoses. A positive diagnosis was obtained in 46.6% and 56.2% of patients with PillCam SB2 and MiroCam capsule, respectively, so that the procedures identified 78.6% and 95.2% of positive cases, respectively (P = .02). Small-bowel transit time and capsule reading time were significantly longer in MiroCam procedures. LIMITATIONS: Technical failures possibly related to capsule interference. CONCLUSION: This study shows at least comparable efficiency of the MiroCam compared with the PillCam SB2 capsule system for the diagnosis of obscure GI bleeding.

High diagnostic and clinical impact of small-bowel capsule endoscopy in patients with hereditary hemorrhagic telangiectasia with overt digestive bleeding and/or severe anemia.

BACKGROUND: Patients with hereditary hemorrhagic telangiectasia (HHT) often present with recurrent anemia because of epistaxis or GI bleeding in relation to telangiectases mostly located in the stomach or small bowel. Capsule endoscopy is considered a major diagnostic tool for small-bowel diseases, but the impact of capsule endoscopy imaging on patient management in HHT is poorly understood. OBJECTIVE: To clarify the contribution of capsule endoscopy in selected patients with HHT. DESIGN: Prospective, descriptive study. SETTING: Multicenter, two university hospital tertiary-care centers, from January 2003 to June 2007. PATIENTS: This study involved 30 patients with HHT and severe anemia (hemoglobin <9 g/dL; normal: 11-15 g/dL) and minimal epistaxis or moderate anemia but overt GI bleeding. INTERVENTION: Capsule endoscopy investigation. MAIN OUTCOME MEASUREMENTS: Clinical characteristics and capsule endoscopy results and their clinical consequences. RESULTS: Capsule endoscopy detected gastric and small-bowel telangiectases in 14 (46.7%) and 26 (86.7%) cases, respectively. Active bleeding was present in 36.7% of cases. Diffuse telangiectases were detected in 42.3% without correlation with age, sex, or type of HHT mutation. Further investigations were carried out as a consequence of the capsule endoscopy results in 67% of cases. Treatment, consisting mostly of endoscopic argon plasma coagulation, was scheduled in 46.7% of patients. LIMITATIONS: Our population was essentially composed of patients with the ALK1 mutation. CONCLUSION: This study shows that there is a high diagnostic yield for capsule endoscopy in selected patients with HHT. Capsule endoscopy makes possible precise mapping of lesions and has a considerable impact on the management of these selected patients by using a predefined algorithm: a limited number of accessible lesions is suitable for endoscopic treatment, whereas innumerable diffuse lesions require a medical approach. We suggest that capsule endoscopy could be a first-line, noninvasive, digestive tract examination in selected patients with HHT.

Revisiting the immunophenotype of nephrogenic adenoma.

Nephrogenic adenoma (NA) is a rare benign lesion of the urinary tract. Although its histogenesis is still debated, several reports suggest that the lesion has a renal tubular cell origin or differentiation. As NA can be difficult to distinguish from malignant conditions such as prostate cancer, there is a need for reliable markers. Unfortunately, it has been reported that NA cells also stained positive for the prostate cancer marker alpha-methylacyl-coenzyme A racemase (AMACR). Because all the previous studies have used an avidin-biotin (AB) detection procedure, and because cells with tubular renal differentiation are likely to contain a high level of endogenous biotin, we investigated in NA the expression of several markers including AMACR, using both AB and biotin-free detection systems. We assessed the expression of p63, cytokeratins 7 and 20, CD10 (proximal tubule marker), MUC1 (distal tubule marker), PAX2, and AMACR on 14 NAs (from 6 patients) grouped on a tissue microarray. The tissue microarray also included renal, urothelial, and prostate tissues. Staining was detected using both AB and biotin-free Envision systems. Detection with the AB procedure leads to nonspecific staining in kidney samples and NA. More specific expression was obtained by using the Envision kit, and only CK7, PAX2, and MUC1 remained positive in NA, without any AMACR staining. These findings provide supporting evidence that NA has the differentiation of distal renal tubules, and strongly suggest that AMACR, when detected with a biotin-free procedure, can be used as a reliable marker for distinguishing NA from prostate cancer.

Micellisation and immunoreactivities of dimeric beta-caseins.

Bovine beta-casein (beta-CN) is a highly amphiphilic micellising phospho-protein showing chaperone-like activity in vitro. Recently, existence of multiple sequential epitopes on beta-CN polypeptide chain in both hydrophilic-polar (psi) and hydrophobic-apolar domains (phi) has been evidenced. In order to clarify specific contribution of polar and apolar domains in micellisation process and in shaping immunoreactivity of beta-CN, its dimeric/bi-amphiphilic "quasi palindromic" forms covalently connected by a disulfide bond linking either N-terminal (C4 beta-CND) or C-terminal domain (C208 beta-CND) were produced and studied. Depending on the C- or N-terminal position of inserted cysteine, each dimeric beta-CN contains one polar/apolar region at the centre and two external hydrophobic/hydrophilic ends. Consequently, such casein dimers have radically different polarities/hydrophobicities on their outside surfaces. Dynamic light scattering (DLS) measurements indicate that these dimeric casein molecules form micelles of different sizes depending on arrangement of polar fragments of the beta-CN mutants in their constrained dimers. Non-aggregated dimers have different hydrodynamic diameters that could be explained by their different geometries. Measurements of fluorescence showed more hydrophobic environment of Trp residues of C208 beta-CND, while in similar experimental conditions Trp residues of C4 beta-CND and native beta-CN were more exposed to the polar medium. Both fluorescence and DLS studies showed greater propensity for micellisation of the dimeric beta-CNs, suggesting that the factors inducing the formation of micelles are stronger in the bi-amphiphilic dimers. 1-anilino-naphthalene-8-sulfonate (ANS) binding studies showed different binding of ANS by these dimers as well as different exposition of ANS binding (hydrophobic) regions in the micellar states. The differences in fluorescence resonance energy transfer (FRET) profiles of C4 beta-CND and C208 beta-CND can be explained by differences of distances and/or by differences of relative orientations of the donor (Trp) and acceptor (ANS), as well as by differences in quenching properties of the disulfide bridges and intra-molecular hydrophobic interactions. The immunoreactivity assays showed somewhat lower IgE response to C208 beta-CND than to C4 beta-CND. Thus, dimerization of C208 beta-CN, connecting two C-terminal hydrophobic domains of two monomers doubling long-range hydrophobic interactions, possibly may hide a part of epitopes in the hydrophobic interface/core of C208 beta-CND that is consistent with the results of DLS and fluorescence studies. The obtained results indicate structural differences of dimers - possibly the formation of Y- and U-shaped structures for C208 beta-CND and C4 beta-CND, respectively. This study not only demonstrated the importance of the organization of polar and hydrophobic regions during micellisation of the constrained and oriented beta-CN dimers but also confirmed a possible role of C-terminal hydrophobic domain in the immunoreactivity profile of native beta-CN.

Effects of heating and glycation of beta-lactoglobulin on its recognition by IgE of sera from cow milk allergy patients.

beta-Lactoglobulin (beta-LG) is one of the cow's major milk proteins and the most abundant whey protein. This globular protein of about 18 kDa is folded, forming a beta-barrel (or calyx) structure. This structure is stabilized by two disulfide bonds and can be altered by heating above 65 degrees C. beta-LG is also one of the major allergens in milk. Heating is one of the most common technologic treatments applied during many milk transformations. During heating in the presence of reducing sugars, beta-LG is also submitted to the Maillard reaction, which at the first stage consists of the covalent fixation of sugars on the epsilon-amino groups of lysyl residues. The following steps are condensation and polymerization reactions leading to the formation of melanoidins (brown pigments). Despite the frequency of use of heating during milk transformation, the effects of heat-induced denaturation and of glycation of beta-LG on its recognition by IgE from cow's milk allergy (CMA) patients are not fully understood. The objectives of our work were to evaluate the effect of heat-induced denaturation of bovine beta-LG on binding of IgE from CMA patients and to determine the effect of moderate glycation on the degree of recognition by IgE. We showed that heat-induced denaturation (loss of tertiary and secondary structures) of beta-LG is associated with weaker binding of IgE from CMA patients. It was also shown that moderate glycation of beta-LG in early stages of Maillard reaction has only a small effect on its recognition by IgE, whereas a high degree of glycation has a clear "masking" effect on the recognition of epitopes. This demonstrates the importance of epsilon-amino groups of lysines in the definition of epitopes recognized by IgE.

Engineering of caseins and modulation of their structures and interactions.

Beta-casein (beta-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air-water interface are not well known. In order to study how the forced dimerisation influences functional properties of beta-CN, recombinant wild-type beta-CN was produced and distal cysteinylated forms of recombinant beta-CN were engineered. We show that 1) cysteinylated beta-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air-water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of beta-CN.