Burkhard Fleckenstein

The Molecular Basis for Oat Intolerance in Patients with Celiac Disease

ABSTRACT Background Celiac disease is a small intestinal inflammatory disorder characterized by malabsorption, nutrient deficiency, and a range of clinical manifestations. It is caused by an inappropriate immune response to dietary gluten and is treated with a gluten-free diet. Recent feeding studies have indicated oats to be safe for celiac disease patients, and oats are now often included in the celiac disease diet. This study aimed to investigate whether oat intolerance exists in celiac disease and to characterize the cells and processes underlying this intolerance. Methods and Findings We selected for study nine adults with celiac disease who had a history of oats exposure. Four of the patients had clinical symptoms on an oats-containing diet, and three of these four patients had intestinal inflammation typical of celiac disease at the time of oats exposure. We established oats-avenin-specific and -reactive intestinal T-cell lines from these three patients, as well as from two other patients who appeared to tolerate oats. The avenin-reactive T-cell lines recognized avenin peptides in the context of HLA-DQ2. These peptides have sequences rich in proline and glutamine residues closely resembling wheat gluten epitopes. Deamidation (glutamine→glutamic acid conversion) by tissue transglutaminase was involved in the avenin epitope formation. Conclusions We conclude that some celiac disease patients have avenin-reactive mucosal T-cells that can cause mucosal inflammation. Oat intolerance may be a reason for villous atrophy and inflammation in patients with celiac disease who are eating oats but otherwise are adhering to a strict gluten-free diet. Clinical follow-up of celiac disease patients eating oats is advisable.

HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions. We used intestinal T cells from celiac patients to map DQ2 and DQ8 epitopes within 2 representative gluten proteins, alpha-gliadin AJ133612 and gamma-gliadin M36999. For alpha-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of 2 separate regions. For gamma-gliadin, DQ2- and DQ8-restricted T cells recognized deamidated peptides of the same region. Some gamma-gliadin peptides were recognized by T cells in the context of DQ2 or DQ8 when bound in exactly the same registers, but with different requirements for deamidation; deamidation at peptide position 4 (P4) was important for DQ2-restricted T cells, whereas deamidation at P1 and/or P9 was important for DQ8-restricted T cells. Peptides combining the DQ2 and DQ8 signatures could be presented by DQ2, DQ8, and trans-encoded DQ heterodimers. Our findings shed light on the basis for the HLA associations in celiac disease and type 1 diabetes.

Refining the Rules of Gliadin T Cell Epitope Binding to the Disease-Associated DQ2 Molecule in Celiac Disease: Importance of Proline Spacing and Glutamine Deamidation

Celiac disease is driven by intestinal T cells responsive to proline-rich gluten peptides that often harbor glutamate residues formed by tissue transglutaminase-mediated glutamine conversion. The disease is strongly associated with the HLA variant DQ2.5 (DQA1*05, DQB1*02), and intestinal gluten-reactive T cells from DQ2.5-positive patients are uniquely restricted by this HLA molecule. In this study, we describe the mapping of two novel T cell epitopes of γ-gliadin and the experimental identification of the DQ2.5 binding register of these and three other γ-gliadin epitopes. The new data extend the knowledge base for understanding the binding of gluten peptides to DQ2.5. The alignment of the experimentally determined binding registers of nine gluten epitopes reveal positioning of proline residues in positions P1, P3, P6, and P8 but never in positions P2, P4, P7, and P9. Glutamate residues formed by tissue transglutaminase-mediated deamidation are found in position P1, P4, P6, P7, or P9, but only deamidations in positions P4 and P6, and rarely in P7, seem to be crucial for T cell recognition. The majority of these nine epitopes are recognized by celiac lesion T cells when presented by the related but nonassociated DQ2.2 (DQA1*0201, DQB1*02) molecule. Interestingly, the DQ2.2 presentation for most epitopes is less efficient than presentation by the DQ2.5 molecule, and this is particularly prominent for the α-gliadin epitopes. Contrary to previous findings, our data do not show selective presentation of DQ2.5 over DQ2.2 for gluten epitopes that carry proline residues at the P3 position.

Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies.

Isozyme‐specific antibodies were raised against peptides from the low‐homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double‐labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co‐localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system.

Redox Regulation of Transglutaminase 2 Activity

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys230, Cys370, and Cys371 that is involved in oxidative inactivation of TG2. Within this triad, Cys370 was found to participate in disulfide bonds with both Cys230 and its neighbor, Cys371. Notably, Ca2+ was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys230 appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys370–Cys371 through formation of the Cys230–Cys370 disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys230 is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

Antigen Presentation to Celiac Lesion-Derived T Cells of a 33-Mer Gliadin Peptide Naturally Formed by Gastrointestinal Digestion

Celiac disease is an HLA-DQ2-associated disorder characterized by intestinal T cell responses to ingested wheat gluten proteins. A peptide fragment of 33 residues (α2-gliadin 56–88) produced by normal gastrointestinal proteolysis contains six partly overlapping copies of three T cell epitopes and is a remarkably potent T cell stimulator after deamidation by tissue transglutaminase (TG2). This 33-mer is rich in proline residues and adopts the type II polyproline helical conformation in solution. In this study we report that after deamidation, the 33-mer bound with higher affinity to DQ2 compared with other monovalent peptides harboring gliadin epitopes. We found that the TG2-treated 33-mer was presented equally effectively by live and glutaraldehyde-fixed, EBV-transformed B cells. The TG2-treated 33-mer was also effectively presented by glutaraldehyde-fixed dendritic cells, albeit live dendritic cells were the most effective APCs. A strikingly increased T cell stimulatory potency of the 33-mer compared with a 12-mer peptide was also seen with fixed APCs. The 33-mer showed binding maximum to DQ2 at pH 6.3, higher than maxima found for other high affinity DQ2 binders. The 33-mer is thus a potent T cell stimulator that does not require further processing within APC for T cell presentation and that binds to DQ2 with a pH profile that promotes extracellular binding.

Cutting Edge: Link Between Innate and Adaptive Immunity: Toll-Like Receptor 2 Internalizes Antigen for Presentation to CD4+ T Cells and Could Be an Efficient Vaccine Target

An ideal vaccine for induction of CD4+ T cell responses should induce local inflammation, maturation of APC, and peptide loading of MHC class II molecules. Ligation of Toll-like receptor (TLR) 2 provides the first two of these three criteria. We have studied whether targeting of TLR2 results in loading of MHC class II molecules and enhancement of CD4+ T cell responses. To dissociate MHC class II presentation from APC maturation, we have used an antagonistic, mouse anti-human TLR2 mAb (TL2.1) as ligand and measured proliferation of a mouse Cκ-specific human CD4+ T cell clone. TL2.1 mAb was 100-1000 times more efficiently presented by APC compared with isotype-matched control mAb. Moreover, TL2.1 mAb was internalized into endosomes and processed by the conventional MHC class II pathway. This novel function of TLR2 represents a link between innate and adaptive immunity and indicates that TLR2 could be a promising target for vaccines.

Evaluation of an optically active crown ether for the chiral separation of di- and tripeptides

The direct optical resolution of a number of di- and tripeptides was achieved by capillary zone electrophoresis using an enantioselective crown ether as buffer additive. The protonated primary amines form inclusion complexes with the crown ether. Chiral resolution is based on different stability constants of the diastereomeric complexes thereby changing the electrophoretic mobilities of the enantiomers. Enantioselectivity is strongly affected by the distance between the amine functionality and the chiral carbon atom. This effect was studied using di- and tripeptides especially synthesized for this purpose. In general, baseline resolution was obtained for those peptides with the amine group located as far as four bonds from the stereogenic center. Additionally, tripeptides possessing two chiral centers were separated to investigate the potential of the chiral selector for the analysis of complex analytes with related structures. Experimental factors such as crown ether concentration, buffer pH and temperature also show a strong influence on the resolution. These factors can be successfully employed for method optimization.

Protection against measles virus encephalitis by monoclonal antibodies binding to a cystine loop domain of the H protein mimicked by peptides which are not recognized by maternal antibodies.

After immunization with measles virus (MV) several monoclonal antibodies (MAbs) were obtained, which reacted with peptides corresponding to the amino acids 361-410 of the haemagglutinin protein (MV-H). Three of these MAbs (BH6, BH21 and BH216) inhibited haemagglutination, neutralized MV in vitro and protected animals from a lethal challenge of rodent-adapted neurotropic MV. These MAbs reacted with the 15-mer peptides H381 and H386 defining their overlapping region 386-395 as a sequential neutralizing and protective epitope, which can be imitated by a short peptide. H381 and H386 share two Cys residues (C(386)KGKIQALC(394)ENPEWA) and for optimal MAb binding of peptide (or MV) disulphide bonds were required in addition to a linear C-terminal extension. Other MAbs bound to peptides C- (BH147, BH195) and N-terminally (BH 168, BH 171) adjacent to the loop but did not neutralize or protect. When sera from measles patients or from women of child-bearing age were tested with the peptides corresponding to this haemagglutinating and neutralizing epitope (HNE), none of the sera recognized the 15-mer peptides of this region, while some reactivity was found to 30-mers homologous to different wild-type mutants. Its lack of recognition by maternal antibodies and its high degree of conservation would make the HNE loop an attractive candidate to include into a subunit vaccine, which could be administered during early childhood, independent of immune status.

Antibodies to a new linear site at the topographical or functional interface between the haemagglutinin and fusion proteins protect against measles encephalitis

The haemagglutinin protein (H) of measles virus (MV) binds to susceptible cells and collaborates with the fusion protein (F) to mediate fusion of the virus with the cell membrane. Binding and fusion activity of the virus can be monitored by haemagglutination and haemolysis, respectively, of monkey erythrocytes. Most monoclonal antibodies (MAbs) with haemolysis inhibiting activity (HLI+) are either MV-F specific and do not inhibit haemagglutination (HI-), or they bind to MV-H and are HI+ by interfering with virus binding. We describe here a small panel of H-specific MAbs (BH47, BH59, BH103, BH129) which bind to a new linear neutralizing epitope, H244-250 (SELSQLS; NE domain), and which prevent virus-cell fusion (HLI+) but not virus binding (HI-). These antibodies also protect against MV encephalitis in an animal model. They do not compete with an HLI+/HI+ antibody (BH216) which binds to the haemagglutinin noose epitope (HNE). The antibodies described here and the HNE-specific antibodies are functionally distinct and define two topographically non-overlapping interfaces, supposedly with a bias towards the host cell MV-receptor and the fusion protein respectively. The proximity of the CD46 downregulating amino acid Arg-243 may suggest a functional link between the domain described here and the CD46 binding domain. This new protective linear site is also of potential interest for the design of a subunit-based vaccine.