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Ian R Mackay

Publications and source records attributed to Ian R Mackay.

At least 19 recordsLinked to original sources
PubMed2006

Molecular characterization of a disease associated conformational epitope on GAD65 recognised by a human monoclonal antibody b96.11.

Autoantibodies to the 65kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65. Recent studies indicate that b96.11 antibody specificity predicts the risk of developing type 1 diabetes in prediabetic individuals. We describe the use homology modelling, protein-protein docking simulations and biopanning of random peptide phage displayed libraries with b96.11 to predict contact amino acids on the interface of GAD65/Fab b96.11 complex. Further analysis by in vitro mutagenesis of GAD65 followed by radioimmunoprecipitation refined the amino acids contributing to the b96.11 epitope. Our studies show an interface characterized by a protruding antibody-combining site centered on the long heavy chain CDR3 loop of Fab b96.11 establishing interactions with the critical residue Phe(344) in the core of the epitope on GAD65, surrounded by charged sites within (375)RK(376) and (305)DER(307). The epitope requires residues from both middle and the C-terminal domains, and is the first precise definition of an epitope on GAD65. The nature of the b96.11 epitope leads to considerations of potential structural variations for differences in antigenicity between the isoforms GAD65 and GAD67. The study shows the utility of using a combination of in silico techniques and experimental data for molecular characterization and localization of conformational epitopes for which crystal structures are lacking.

Amino Acid Sequence↗
PubMed2006

IL-2 receptor alpha deficiency and features of primary biliary cirrhosis.

Congenital immune deficiency states have often been valuable experimental models of nature that have significantly enhanced our understanding of the immune response. The relationship between CD4+, CD25+ and Treg cells in the induction of autoimmunity has attracted significant attention. We report herein a male child of consanguineous parents who developed at six months recurrent infections, and at age 5 years, liver dysfunction with serological expression of primary biliary cirrhosis (PBC), an autoimmune liver disease that usually affects middle-aged women. Histologically, there was intense mononuclear cell lymphoid infiltration of the intrahepatic portal tracts, CD3+CD4+T cell lymphopenia in blood and serum antibody to PDC-E2. Peripheral blood lymphocytes were completely deficient of the alpha subunit of the IL-2 receptor (IL-2Ralpha, CD 25), a marker for regulatory T cells (Tregs). Allogenic stem cell transplantation led to full recovery. This case illustrates the role of deficiency of CD4+CD25+ Treg cells in causing autoimmunity, and speaks to the potential use of allogenic stem cell transplantation for immunoreconstitution in adult PBC.

Autoimmunity↗
PubMed2006

Anti-mitochondrial antibodies and primary biliary cirrhosis in TGF-beta receptor II dominant-negative mice.

Primary biliary cirrhosis (PBC) is an autoimmune disease of the liver, characterized by lymphocytic infiltrates in portal tracts, selective destruction of biliary epithelial cells, and anti-mitochondrial Abs (AMAs). The elucidation of early events in the induction of tissue inflammation and autoimmunity in PBC has been hampered by the cryptic onset of the disease, the practical limitations in accessing the target tissue, and the lack of a suitable animal model. We demonstrate in this study that a mouse transgenic for directed expression of a dominant-negative form of TGF-beta receptor type II (dnTGFbetaRII), under the direction of the CD4 promoter, mimics several key phenotypic features of human PBC, including spontaneous production of AMAs directed to the same mitochondrial autoantigens, namely PDC-E2, BCOADC-E2, and OGDC-E2. The murine AMAs also inhibit PDC-E2 activity. Moreover, there is lymphocytic liver infiltration with periportal inflammation analogous to the histological profile in human PBC. Additionally, the serum cytokine profile of affected mice mimics data in human PBC. The concomitant presence of these immunopathological features in the transgenic mice suggests that the TGF-betaRII pathway is implicated in the pathogenesis of PBC. Finally, these data point away from initiation of autoimmunity by mechanisms such as molecular mimicry and more toward activation of an intrinsically self-reactive T cell repertoire in which necessary regulatory T cell influences are lacking.

Animals↗
PubMed2006

Identification of 2-nonynoic acid, a cosmetic component, as a potential trigger of primary biliary cirrhosis.

Antimitochondrial antibodies (AMA) are unique among autoimmune serologic reactants because of their extremely high association with the index disease primary biliary cirrhosis (PBC). This autoantibody response is specifically directed only to the lipoyl domain of the mitochondrial 2-oxo-acid dehydrogenase complexes, which prompted us to search for environmental mimotopes in the form of xenobiotics and led to our identification of 2-octynoic acid as a high-affinity reactant for AMA. To focus on the chemical characteristics requisite for binding of AMA to the xenobiotic-modified self-peptide, quantitative structure-activity relationship (QSAR) studies were performed using a panel of alkynoic compounds, including examination of the length of the carbon chain and the location of the triple bond in the identified mimotope. Analyses of octynamides that varied in the position of the triple bond demonstrated that only the 2-octynamide reacted strongly with PBC sera. Furthermore, among 2-alkynamides with varying carbon chain length, 2-octyn-, 2-nonyn- (particularly) and 2-decynamide exhibited the highest reactivity. Thus, an optimal chemical structure of the xenobiotically modified epitope recognized by AMA-positive PBC sera is provided by 2-nonynoic acid. The methyl ester of this compound is ranked 2,324th out of 12,945 compounds to which there is occupational exposure, with an 80% female prevalence due to its use in cosmetic products. Our findings illustrate an unusual polyreactivity of anti-PDC-E2 and support the idea of epitope mimicry in the genesis of this autoantibody and perhaps of PBC itself.

Autoantibodies↗
PubMed2006

NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti-PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9-10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

Adoptive Transfer↗
PubMed2006

Characterisation of an autoreactive conformational epitope on GAD65 recognised by the human monoclonal antibody b78 using a combination of phage display, in vitro mutagenesis and molecular modelling.

Autoantibodies to the diabetes autoantigen, the 65kDa isoform of glutamic acid decarboxylase (GAD65), react with conformational epitopes defined according to linear sequences but not according to structural information, or contact sites with the antibody paratope. To ascertain such information for an exemplary human monoclonal antibody (mAb) to GAD65, b78, we combined antibody screening of phage-displayed peptide libraries, alanine mutagenesis of selected motifs, homology modelling of the PLP and C-terminal regions of GAD65, and molecular dynamics to examine for structural effects of mutagenesis. By phage display, mAb b78 selected phagotopes containing acidic residues (D, E), hydrophobic residues (Y, F or W) and LRS that localised to a possible surface-exposed conformational epitope on the combined homology model. Alanine mutants of GAD65 based on deduced contact residues were examined for binding with b78 and control sera. Mutation of (524)SRL(526), (572)DF(573) and (498)KPQ(500) reduced reactivity of b78 with mutant GAD65 > 50%. Molecular dynamics indicated that mutation of (498)KPQ(500) caused structural changes that could account for effects of this mutation. Thus phage display in combination with molecular modelling identified contact residues within a highly conformational epitope for mAb b78 in the C-terminus of GAD65. These techniques should have broad applicability to definition of epitope structure.

Amino Acid Sequence↗
PubMed2006

IL-2 receptor alpha(-/-) mice and the development of primary biliary cirrhosis.

Recently, we identified a child born with a genetic deficiency of IL-2 receptor alpha (IL-2Ralpha, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2Ralpha/CD25 deficient (IL-2Ralpha(-/-)) mice and wild-type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2Ralpha(-/-), but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4(+) and CD8(+) T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-gamma, TNF-alpha, IL-2 and IL-12p40. Of importance is the finding that the IL-2Ralpha(-/-) mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2Ralpha(-/-) mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage.

Animals↗
PubMed2005

Amino acids critical for binding of autoantibody to an immunodominant conformational epitope of the pyruvate dehydrogenase complex subunit E2: identification by phage display and site-directed mutagenesis.

The E2 subunit of the mitochondrial multienzyme pyruvate dehydrogenase complex (PDC-E2) is the major autoantigen in the liver disease, primary biliary cirrhosis (PBC). An epitope region which has been localized to amino acids 91-227 is believed to include the residue K173 to which is attached the lipoyl cofactor. We investigated structural features of this epitope region by screening random peptide phage-displayed libraries and identified prevalent phagotopes that contained likely contact amino acids in separate regions of the linear sequence, H132M133, and F178, V180. These were confirmed by site-directed alanine mutagenesis singly or in combination of the HM and FV residues in wild-type (wt) PDC-E2, and by immunization of rabbits with phage that expressed peptides MHLNTPP or FVLPWRI. The lipoyl lysine K173 also was mutated. Reactivities of mutants and wild-type (wt) PDC-E2, compared by ELISA using 12 PBC sera, showed decremental reactivity of mutant versus wt PDC-E2 (normalized to 100%): wt PDC-E2 (100%)>>PDC-E2(F178A,V180A) (mean+/-S.D., 59+/-17%)>PDC-E2(M133A) (50+/-13%)>PDC-E2(H132A) (36+/-13%)>PDC-E2(H132A,M133A) (28+/-8%)>PDC-E2(H132A,M133A,F178V,M180A) (18+/-13%). Notably PDC-E2(K173A) retained full reactivity (93+/-21%). Rabbits immunized with phage peptides generated antibodies reactive with entire PDC-E2. Our data convincingly validate phage library technology for defining spatially disparate contact residues for conformational epitopes. Ensuing data could be generally applicable to search for occult extrinsic agents as initiators of autoimmunity.

Amino Acid Sequence↗
PubMed2005

Requirement of multiple phage displayed peptide libraries for optimal mapping of a conformational antibody epitope on CCR5.

In the absence of information from crystallography, conformational epitopes can often be discerned by antibody screening of phage displayed random peptide libraries. However the context in which the peptide is displayed, and the number of copies displayed in the library, can influence results and interpretations. Here, the monoclonal antibodies 3A9 specific for the transmembrane chemokine receptor CCR5, and CII-C1 specific for type II collagen, were used to screen multiple phage-displayed peptide libraries in which peptides were displayed in either the pIII or pVIII coat proteins. ELISA was used to test for reactivity and cross-inhibitory activity of isolated phage clones. Based on sequences of reactive phage inserts, epitope motifs were initially inferred from a molecular model of CCR5 and subsequently confirmed experimentally using mutagenesis to alanine. For each mAb, phage sequences from pIII biopannings were more diverse than from pVIII biopannings. Notably, sequences from either biopanning were cross-inhibitory despite a lack of linear sequence homology. For CCR5, residues 88H and 94W in the first loop of CCR5 were identified by pIII biopannings, and 7S9IYD11 at the N-terminus by pVIII biopannings. Thus conformational epitopes can be identified using phage display, but optimal mapping of complex epitopes can require the use of multiple peptide libraries.

Amino Acid Motifs↗
PubMed2005

Arthritogenic anti-type II collagen antibodies are pathogenic for cartilage-derived chondrocytes independent of inflammatory cells.

OBJECTIVE: Some monoclonal antibodies (mAb) to type II collagen (CII) are arthritogenic upon passive transfer to mice. We undertook this study to investigate whether such mAb are pathogenic in the absence of mediators of inflammation. METHODS: The arthritogenic mAb CIIC1 and M2139, and the nonarthritogenic mAb CIIF4, each reactive with a distinct and well-defined conformational epitope on CII, were compared with control mAb GAD6. Bovine chondrocytes were cultured with one of the mAb, and on days 3, 6, and 9, antibody binding by chondrocytes and newly synthesized extracellular matrix (ECM) was examined by immunofluorescence, morphologic effects were studied by electron microscopy, and synthesis of matrix components was determined by metabolic labeling with (3)H-proline for collagen and (35)S-sulfate for proteoglycans. RESULTS: All 3 mAb to CII bound to the matrix. CIIC1 and M2139 adversely affected the cultures, whereas CIIF4 did not. CIIC1 caused disorganization of CII fibrils in the ECM without affecting chondrocyte morphology, and increased matrix synthesis. M2139 caused thickening and aggregation of CII fibrils in the ECM and abnormal chondrocyte morphology but matrix synthesis was unaffected. CONCLUSION: The unique arthritogenic capacity of particular anti-CII mAb upon passive transfer could be explained by their adverse, albeit differing, effects in primary cultures of chondrocytes. Such effects occur independent of inflammation mediators and are related to the epitope specificity of the mAb. Interference with the structural integrity of CII could precede, and even initiate, the inflammatory expression of disease.

Animals↗
PubMed2005

From bases to basis: linking genetics to causation in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a multifactorial autoimmune disease with inherited and environmental components in pathogenesis. It is exceptional among autoimmune diseases in showing strong heritability according to familial occurrence and monozygotic twins concordance, yet with weak associations with the usual genetic risk elements for autoimmunity, such as the HLA alleles. Among the latter, there is risk (at least in some populations) conferred by HLA DRB1*08 and possibly some protection by DRB1*11. However, the inconsistency among studies on HLA is surprising, given that PBC is a relatively homogenous disease entity. Among non-HLA genes, some studies implicate polymorphisms of genes for cytotoxic T-lymphocyte antigen-4, interleukin-2, or interleukin-10; polymorphisms of the vitamin D receptor could synergize with low sunlight exposure to create deficiency of the immunoregulatory factor, activated vitamin D. A new lead is available from the finding in female subjects with PBC of an increase in the degree of monosomy of the X chromosome that is presumed to carry immune response genes. A further suggested source of inquiry is the apparent protection of African-American women from PBC. Finally, data on inheritance should be sought in PBC by descent methodology, rather than by cross-sectional association studies in cases and control subjects, and based on analysis of a large number of families with an affected member through a worldwide effort.

Autoimmune Diseases↗
PubMed2005

The etiopathogenesis of autoimmunity.

Acquisition by mammals of an adaptive immune system was an evolutionary leap, but occurred at the cost of autoimmunity. The necessity for self-recognition was appreciated in 1900, but autoimmune disease did not become a clinical reality until the 1950s-still the perimeters are indistinct. Autoimmune responses recapitulate the complex events of normal immune responses but cannot shut down. Immune tolerance is established during repertoire development centrally in thymus or bone marrow by deletion of self-reactive immunocytes, and is supplemented peripherally by regulatory T cells (Tregs). A startling discovery is the autoimmune regulator AIRE gene that enables expression of organ-specific autoantigens for intrathymic deletional tolerance of T cells. The origins, activities, and markers of Tregs are under intensive investigation. Of genes implicated in autoimmune, only few are characterized; contributions of environment are similarly uncertain. Time-latency considerations implicate stochastic factors or chance in the etiopathogenesis of autoimmunity, whether they are somatic mutations or successive random gene-environment interactions. Solutions to etiopathogenesis require novel experimental models and finely designed gene-environment studies on human populations. Perhaps immunomodulatory therapies will effect cures before causes are fully understood.

Animals↗
PubMed2005

Heritability of levels of autoantibodies using the method of plotting regression of offspring on midparent (ROMP).

The genetic control of the levels of autoantibodies has rarely been examined. We examined the heritability of autoantibodies to glutamic acid decarboxylase (GAD65) in type 1 diabetes, and to thyroglobulin (Tg) in chronic lymphocytic thyroiditis and thyrotoxicosis, using regression of offspring on midparent (ROMP) methods. Levels of autoantibodies in patients and their parents were significantly correlated in thyrotoxicosis (R2 = 0.569, p = 0.001), consistent with the reported Gm association, but not in chronic lymphocytic thyroiditis or type 1 diabetes. Extension of the procedure to other autoantibody disorders could be informative.

Adult↗
PubMed2004

Antibodies against the CB10 fragment of type II collagen in rheumatoid arthritis.

Antibodies against intact type II collagen (CII) are a feature of rheumatoid arthritis (RA) but have limited diagnostic value. Here we assess whether either of the two major cyanogen bromide fragments of CII, namely CB10 or CB11, are more sensitive substrates for the detection of antibodies in RA. Cleavage of bovine CII with cyanogen bromide yielded CB10 and CB11; these were purified by column chromatography for use in an enzyme-linked immunosorbent assay. Serum antibodies were measured in patients with RA, psoriatic arthritis (PsA), osteoarthritis (OA) and blood donors. Results were compared with those using intact CII. Antibodies against CB10 were found in as many as 88% of 96 patients with long-standing RA, but only 12% of 33 patients with PsA, 6% of 34 patients with OA and 3% of 93 control sera. Lower frequencies for these diseases were obtained on testing for antibodies against CB11: 50%, 6%, 21% and 2%, respectively. The sensitivity of detection in RA of antibodies against CB10 compared with antibodies against intact CII (88% versus 24%) was not at the expense of specificity, which remained high at 94%. The much higher frequency of antibodies against CB10 in RA than in other rheumatic diseases or control sera indicates that CB10 is clearly a more sensitive substrate than the intact collagen molecule and, combined with other assays (rheumatoid factor, anti-cyclic citrullinated peptide [anti-CCP]), might comprise a panel with a highly reliable predictive value. Moreover, our findings should encourage renewed interest in the role of collagen autoimmunity in the pathogenesis of RA.

Adolescent↗
PubMed2004

Measurement of antibodies to collagen II by inhibition of collagen fibril formation in vitro.

Antibodies to type II collagen (collagen II) are pathogenic in experimental collagen-induced arthritis (CIA) and possibly also in rheumatoid arthritis (RA). Hitherto, results of assays for anti-collagen II have proven to be inconsistent. We tested whether mouse monoclonal antibodies (mAbs) to collagen II inhibit the natural self-assembly of soluble triple-stranded collagen II monomers to form insoluble polymeric fibrils. A spectrophotometric assay of self-assembly was based on change in absorbance at 313 nm, observed over 0-60 min after neutralisation and warming of a solution of monomeric collagen II. Two mAbs to collagen II (CII-CI and M2.139) strongly inhibited self-assembly of collagen II but not collagen I, whereas another antibody, CII-F4, and an irrelevant control mAb did not. Notably, CII-CI and M2.139, but not CII-F4, induce arthritis on passive transfer to naïve mice. The arthritogenic effects of mAbs CII-CI and M2.139 in vivo, and inhibition of collagen II self-assembly in vitro, may be attributable to interference with critical epitopes at sites essential for the stabilisation of the mature polymeric collagen II fibril, and, hence, the integrity of the entire cartilage matrix. This assay for inhibition of self-assembly of collagen II could be developed for routine measurement of anti-collagen II in body fluids as a marker of early RA, and perhaps also to distinguish populations of antibodies to collagen II that either have or lack the capacity to perpetuate arthritis.

Animals↗
PubMed2004

Autoimmune epitopes: autoepitopes.

The identity of reactants for autoantibodies has been successively refined from whole cellular organelles (immunofluorescence), identified molecules (immunoblot; gene expression libraries), epitope regions (truncated cDNAs; peptide scanning) to contact residues, as described here. Most autoantibodies react with conformational epitopes, in which amino acids distant in the linear sequence come into contiguity by protein folding. Identification of contact sites with the antibody paratope requires particular technologies, crystallography, or antibody screening of phage-displayed random peptide libraries. The latter is illustrated by our studies on the autoepitope for anti-PDC-E2 (AMA) in primary biliary cirrhosis (PBC), anti-GAD65 in type 1 diabetes, and anti-C1 of type II collagen in collagen-induced arthritis. More precise definition of the structure of conformational autoepitopes could (a) clarify controversial aspects of autoimmunity including epitope mimicry, epitope spreading, and molecular spatial relationships between B and T cell autoepitopes, and (b) impact on novel diagnostic and therapeutic (vaccine) molecules.

Animals↗