Steffen U. Friis

Purification and characterisation of antigenic gliadins in coeliac disease

Two gliadins, known to be especially antigenic in coeliac disease, were purified to homogeneity by a series of ion-exchange chromatography steps. Their N-terminal amino acid sequences showed minor differences but clearly classified them as gamma-type gliadins. The purified gliadins were further characterised with respect to amino acid composition, molecular mass and E1(1%)cm at 276 nm. Based on these properties it is suggested that one of them is identical to a gamma-type gliadin, earlier characterised by its nucleotide sequence, whereas the other has not previously been described. The purification procedure may form the basis for the development of a more differentiated analysis of circulating antibodies for diagnosis and makes clinical testing of the toxicity of defined gliadin peptides feasible.

The impact of EndoBarrier gastrointestinal liner in obese patients with normal glucose tolerance and in patients with type 2 diabetes

The duodenal‐jejunal bypass sleeve ((DJBS) or EndoBarrier Gastrointestinal Liner) induces weight loss in obese subjects and may improve glucose homeostasis in patients with type 2 diabetes (T2D). To explore the underlying mechanisms, we evaluated postprandial physiology including glucose metabolism, gut hormone secretion, gallbladder emptying, appetite and food intake in patients undergoing DJBS treatment.

Separation of gliadin at pH 3.1 in a polyacrylamide gel suitable for blotting procedures

A polyacrylamide gel electrophoresis system for the separation of gliadin at acidic pH is described. The gel is cast at neutral pH with polymerization in 20 min. Equilibration of the gel to pH 3.1 takes place during the electrophoresis. The gel is highly uniform with good mechanical properties and therefore suitable for blotting procedures.

Coeliac Disease. Pathogenesis and clinical aspects

The present thesis is based on studies performed at Department of Medical Biochemistry & Genetics, Biochemistry Laboratory C, The Panum Institute, University of Copenhagen and Medical Department A/P, Division of Gastroenterology, Rigshospitalet. A part of the clinical studies was carried out at Medical Department F, Copenhagen County Hospital, Gentofte. I am sincerely indebted to professor med.dr. Ove N o r h and professor med.dr. Hans Sjostrom for their encouraging and consistent support. It has been a pleasure and an honour to be a member of their group in Biochemistry Laboratory C, with its high scientific standard and extensive good fellowship. These characteristica can only be achieved by excellent leadership, and a constant input and enthusiasm from all in the staff. The late dr.med. Stig Jarnum, my personal mentor at Rigshospitalet, was always a constructive source of inspiration for me. I often look back on his impressive idealism and commitment, and the very valuable discussions in his office in the late afternoons. The staff at the three institutions are thanked for their generous help. In particular, I highly appreciate the skilhl technical assistance from Dorit Anthonsen, Bodil Petersen and Lise-Lotte Niels-Christiansen. I wish to express my gratitude to professor dr.med. E. Dabelsteen and technician Hanne L. Hansen for their expert help with the immunofluorescence experiments. I acknowledge the valuable contributions from the co-authors (J Boserup, E. Gudmand-Hq~er, SD. Kristensen, K. Larsen, SA! Rasmussen, A! Riidiger and C. Schaj.2r-Nielsen). Cand. scient. G.H. Hansen is sincerely thanked for fruitfkl discussions and the co-operation invery informative experiments (that unfortunately did not result in an individual publication). The volunteer coeliac patients and healthy controls are cordially thanked for their participation in the studies as are the Danish Coeliac Society and committee member CC Brandt-Pedersen for the support.

The prolamin antibody reactivity against hordein polypeptides in sera from patients with coeliac disease.

The antibody reactivity against the barley prolamin, hordein, was investigated by an immunoblotting technique, in sera from patients with untreated coeliac disease, patients with other gastrointestinal diseases and healthy controls. No characteristic hordein polypeptide antibody pattern could be connected to coeliac disease, as observed in a similar study using different fractions of the wheat prolamin, gliadin. Gliadin- and hordein-immunoadsorbent column experiments demonstrated that the prolamin reactivity originates from the same population of antibodies. It is speculated that distinct antigenic epitopes characteristic for untreated coeliac disease, might reside within a N-terminal repeat region of gliadin.

Gamma-type gliadins cause secretion of prostaglandin E2 in patients with coeliac disease

Coeliac disease is induced by polypeptides in the prolamin fraction of wheat, termed gliadin. It has previously been demonstrated that the alpha-, the beta- and the gamma-gliadin fractions contain toxic components and it has furthermore been strongly indicated that alpha-type gliadins are toxic. Due to insufficient protein separation methods there has been no information as to whether also the gamma-type gliadins are injurious in coeliac disease. We have therefore purified one alpha-type (alpha-39) and two gamma-type gliadins (gamma-36 and gamma-47) in a preparative scale by a combination of different ion exchange chromatographies. The purity was analyzed by high performance liquid chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, while the typing was based on determination of N-terminal amino acid sequence. Six patients with coeliac disease in remission were included in the study. Each of the purified gliadins was given by an intestinal perfusion technique to two patients. The perfusion fluid was collected and analyzed for the concentration of prostaglandin E2 (PGE2) as a marker for a toxic effect. All patients reacted with increased PGE2 secretion. For the first time it is clearly demonstrated that gamma-type gliadins are active in coeliac disease.

Immunomicroscopic localization of the 10 000 molecular weight calcium‐binding protein in the human enterocyte

The cellular localization of the 10 000 molecular weight calcium‐binding protein (CaBP or Calbindin‐D) in the small intestinal epithelium of man was investigated by immunofluorescence and immunoelectron microscopy (immunogold staining). Indirect immunofluorescence on frozen sections showed intracellular staining in the enterocyte. The fluorescence was evenly distributed and no significant differences were observed between crypt and villus cells. No staining was found in goblet cells or in the submucosa. Correspondingly, immunogold labeled antibodies were scattered over the cytoplasm of the enterocyte. The terminal region appeared to be the most intensely decorated and the brush border region showed labeling above the background level. No labeling was associated with intracellular membranes or the basolateral membrane.

Erratum to: Cancer occurrence in Danish diabetic patients: duration and insulin effects

The staff of the Editorial Office regret that this paper was published online before the authors’ corrections were implemented. Changes are shown in colour. 1) The correct affiliation for D. R. Witte is listed below. 2) The third sentence of the Results section of the Abstract should read: The cancer incidence rate ratio was higher among patients using insulin, decreasing from 5 at the start of insulin treatment to after 5 years of insulin use. 3) The final sentence of the third paragraph of the ‘Linkage and tabulation’ section of the ‘Methods’ should read: Follow-up in the two diabetic categories was further subdivided by time since inclusion in the register (diabetes duration), and by time since the second insulin purchase (insulin duration), in intervals of length 3, 6, 12 and 24 months, as illustrated in Fig. 2 . 4) In the ‘Methods’ the last paragraph of the ‘Statistical model and analysis’ section entitled ‘Model for the rate ratio’ should read: The rate ratio is shown graphically by duration of diabetes for diabetic patients not taking insulin, as well as for diabetic patients initiating insulin at 0, and 5 years after diagnosis. 5) The URL in reference 15 should read: .

THE IMMUNOLOGICAL RESPONSE TO GLIADIN IN COELIAC DISEASE SEEKS NOT TO BE OF PRIMARY PATHOGENETIC IMPORTANCE

Patients with untreated coeliac disease have circulating antibodies against gliadin. We have previously demonstrated that the gliadin-antibody pattern is dominated by reactivity against a few polypeptides, tentatively identified as gamma-gliadins by their migration in electrophoresis. A varying degree of reactivity to alfa- and beta-gliadin was detected, but the sera did not react with glutenins. It was accordingly hypothesised that the reaction pattern was due to a specific primary reactivity against some gliadin polypeptides, with a secondary immunization with other polypeptides, initiated by epithelial damage and increased intestinal permeability.Gliadin, digested with pepsin and trypsin (PT-gliadin), has been shown to be toxic. In order to further elucidate the connection between gliadin antigenecity and toxicity, the antibody reactivity in coeliac sera against PT-gliadin was investigated by immunoblotting. The enzymatic digestion of gliadin accomplished low molecular weight polypeptides, which notably did not display antibody reactivity.It is therefore suggested that characteristic gliadin antibody pattern in patients with coeliac disease is not of primary pathogenetic importance, but instead correlated to common HLA-antigens of most of the patients.

38 THE PEPTIDE DOMAINS OF AETIOLOGICAL IMPORTANCE IN COELIAC DISEASE MAY RESIDE WITHIN THE N-TERMINAL REGION OF |[gamma]|-GLIADIN

We have previously demonstrated that patients with coeliac disease have a characteristic gliadin antibody pattern dominated by reactivity against a few polypeptides in the γ-fraction. Recent gliadin- and hordein- (the prolamin fractions of wheat and barley) immunoadsorbent studies have shown, that the prolamin reactivity originates from the same population of antibodies. The antibody reactivity against separated hordein polypeptides was therefore investigated by immunoblotting, but a characteristic pattern could not be connected to coellac disease.The combination of these observations on basis of detailed comparison of available amino acid sequences leads to the speculation, that distinct antigenic epitopes characteristic for coeliac disease may reside within the N-terminal region of gliadins.The antibodies in patients with coeliac disease does however not react with a typical α-gliadin expressed by E.coli, which extends the indications that the antigenic epitopes are in the γ-gliadin fraction.A determination of the aetiologically active peptide domains and an elucidation of the initial events of the pathogenesis should nevertheless be based on direct studies of the enterocytes under in vivo resembling conditions.