A comparative study of antibody expressions in primary biliary cirrhosis and autoimmune cholangitis using phage display

Primary biliary cirrhosis (PBC) and autoimmune cholangitis (AIC) are serologic expressions of an autoimmune liver disease affecting biliary ductular cells. Previously we screened a phage-displayed random peptide library with polyclonal IgG from 2 Australian patients with PBC and derived peptides that identified a single conformational (discontinuous) epitope in the inner lipoyl domain of the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the characteristic autoantigen in PBC. Here we have used phage display to investigate the reactivity of PBC sera from 2 ethnically and geographically distinct populations, Japanese and Australian, and the 2 serologic expressions, PBC and AIC. Random 7-mer and 12-mer peptide libraries were biopanned with IgG from 3 Japanese patients with PBC and 3 with AIC who did not have anti-PDC-E2. The phage clones (phagotopes) obtained were tested by capture enzyme-linked immunosorbent assay (ELISA) for reactivity with affinity-purified anti-PDC-E2, and compared with those obtained from Australian patients with PBC. Peptide sequences of the derived phagotopes and sequences derived by biopanning with irrelevant antisera were aligned to develop a guide tree based on physicochemical similarity. Both Australian and Japanese PBC-derived phagotopes were distributed in branches of the guide tree that contained the peptide sequences MH and FV previously identified as part of an immunodominant conformational epitope of PDC-E2, indicating that epitope selection was not influenced by the racial origin of the PBC sera. Biopanning with either PBC or AIC-derived IgG yielded phagotopes that reacted with anti-PDC-E2 by capture ELISA, further establishing that there is a similar autoimmune targeting in PBC and AIC.

Towards the pathogenesis of autoimmune liver disease

There have been recent improvements in the clinical understanding and definition of the major types of autoimmune liver disease. However, still lacking is knowledge of their prevalence and pathogenesis. Three areas of study are in progress in our laboratory. First, in type 1 autoimmune hepatitis, the search continues to identify a liver/disease-specific autoantigenic reactant. Using hepatocyte membrane preparations, immunoblotting has underlined the problem of distinguishing, among multiple reactants, those that may be causally rather than consequentially related to hepatocellular damage. Second, in primary biliary cirrhosis (PBC), the need for population screening to ascertain prevalence and detect preclinical cases can be met by a rapid automated procedure for detection, by specific enzyme inhibition in microtitre wells, of antibody (anti-M2) to the pyruvate dehydrogenase complex E2 subunit (PDC-E2). Third, the structure of the conformational epitope within the inner lipoyl domain of PDC-E2 is being investigated by screening random phage-displayed peptide libraries using PBC sera. This has yielded phage clones in which the sequence of the peptide insert portrays the structure of this epitope, as judged by clustering of PBC-derived sequences to particular branches of a guide-tree that shows relatedness of peptides, and by reactivity of selected phage clones with anti-PDC-E2. Thus phage display identifies a peptide 'mimotope' of the antibody epitope in the inner lipoyl domain of PDC-E2.

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Fine mapping and replication of genetic risk Loci in primary sclerosing cholangitis

Background and aims. Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammation and fibrosis of the bile ducts eventually leading to biliary cirrhosis. Recent genetic studies in PSC have identified associations at 2q13, 2q35, 3p21, 4q27, 13q31 and suggestive association at 10p15. The aim of this study was to further characterize and refine the genetic architecture of PSC. Methods. We analyzed previously reported associated SNPs at four of these non-HLA loci and 59 SNPs tagging the IL-2/IL-21 (4q27) and IL2RA (10p15) loci in 992 UK PSC cases and 5162 healthy UK controls. Results. The most associated SNPs identified were rs3197999 (3p21 (MST1), p = 1.9 × 10 -6, OR A vs G = 1.28, 95% CI (1.16-1.42)); rs4147359 (10p15 (IL2RA), p = 2.6 × 10 -4, OR A vs G = 1.20, 95% CI (1.09-1.33)) and rs12511287 (4q27 (IL-2/IL-21), p = 3.0 × 10 -4, OR A vs T = 1.21, 95% CI (1.09-1.35)). In addition, we performed a meta-analysis for selected SNPs using published summary statistics from recent studies. We observed genome-wide significance for rs3197999 (3p21 (MST1), P combined = 3.8 × 10 -12) and rs4147359 (10p15 (IL2RA), P combined = 1.5 × 10 -8). Conclusion. We have for the first time confirmed the association of PSC with genetic variants at 10p15 (IL2RA) locus at genome-wide significance and replicated the associations at MST1 and IL-2/IL-21 loci in a large homogeneous UK population. These results strongly implicate the role of IL-2/IL2RA pathway in PSC and provide further confirmation of MST1 association. © Informa Healthcare.

Primary alkaline battery cathodes a three-scale model

A mathematical model for the galvanostatic discharge and recovery of porous, electrolytic manganese dioxide cathodes, similar to those found within primary alkaline batteries is presented. The phenomena associated with discharge are modeled over three distinct size scales, a cathodic (or macroscopic) scale, a porous manganese oxide particle (or microscopic) scale, and a manganese oxide crystal (or submicroscopic) scale. The physical and chemical coupling between these size scales is included in the model. In addition, the model explicitly accounts for the graphite phase within the cathode. The effects that manganese oxide particle size and proton diffusion have on cathodic discharge and the effects of intraparticle voids and microporous electrode structure are predicted using the model.

Exploring the influence of a science content course incorporating explicit nature of science and argumentation instruction on preservice primary teachers' views of nature of science

There exists a general consensus in the science education literature around the goal of enhancing students. and teachers. views of nature of science (NOS). An emerging area of research in science education explores NOS and argumentation, and the aim of this study was to explore the effectiveness of a science content course incorporating explicit NOS and argumentation instruction on preservice primary teachers. views of NOS. A constructivist perspective guided the study, and the research strategy employed was case study research. Five preservice primary teachers were selected for intensive investigation in the study, which incorporated explicit NOS and argumentation instruction, and utilised scientific and socioscientific contexts for argumentation to provide opportunities for participants to apply their NOS understandings to their arguments. Four primary sources of data were used to provide evidence for the interpretations, recommendations, and implications that emerged from the study. These data sources included questionnaires and surveys, interviews, audio- and video-taped class sessions, and written artefacts. Data analysis involved the formation of various assertions that informed the major findings of the study, and a variety of validity and ethical protocols were considered during the analysis to ensure the findings and interpretations emerging from the data were valid. Results indicated that the science content course was effective in enabling four of the five participants. views of NOS to be changed. All of the participants expressed predominantly limited views of the majority of the examined NOS aspects at the commencement of the study. Many positive changes were evident at the end of the study with four of the five participants expressing partially informed and/or informed views of the majority of the examined NOS aspects. A critical analysis of the effectiveness of the various course components designed to facilitate the development of participants‟ views of NOS in the study, led to the identification of three factors that mediated the development of participants‟ NOS views: (a) contextual factors (including context of argumentation, and mode of argumentation), (b) task-specific factors (including argumentation scaffolds, epistemological probes, and consideration of alternative data and explanations), and (c) personal factors (including perceived previous knowledge about NOS, appreciation of the importance and utility value of NOS, and durability and persistence of pre-existing beliefs). A consideration of the above factors informs recommendations for future studies that seek to incorporate explicit NOS and argumentation instruction as a context for learning about NOS.