Mark A. Myers

Pancreatic Islet Cell Cytoplasmic Antibody in Diabetes is Represented by Antibodies to Islet Cell Antigen 512 and Glutamic Acid Decarboxylase

The presence of serum islet cell cytoplasmic antibodies (ICAs) is a standard autoimmune marker for insulin-dependent diabetes mellitus (IDDM). The antigenic molecule(s) responsible for ICA has not been identified, although antibodies to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) do contribute. We tested 129 IDDM sera for antibodies to ICA512 (anti-ICA512), antibodies to GAD (anti-GAD), and ICAs; we tested for inhibition of ICAs with purified recombinant ICA512 and sheep brain GAD; and we tested for immunofluorescence reactivity on COS7 cells transfected with cDNA clones encoding ICA512 and GAD65. The results were that anti-ICA512 antibodies contribute to ICA reactivity and that these, in combination with anti-GAD antibodies, account for most ICA reactivity in IDDM. Anti-ICA512 antibodies were present at a frequency of 51% in 61 patients with early-onset IDDM (age of onset ≤20 years) of short duration (≤1 month) but only in 9% of 68 patients with an onset age of >20 years and/or a disease duration of >1 month. The frequency of anti-GAD antibodies in these sera was similar irrespective of duration or age of onset. Anti-ICA512 and anti-GAD antibodies were demonstrable by indirect immunofluorescence on transfected COS7 cells, and ICA could be inhibited using either recombinant ICA512 or purified brain GAD. We conclude that anti-ICA512 and anti-GAD antibodies contribute to ICA reactivity and that anti-ICA512 antibodies account for the increased frequency of ICA reactivity in early-onset IDDM of short duration.

The peculiar autoimmunity of primary biliary cirrhosis.

Summary: Autoantibodies to mitochondria (AMA, anti‐M2) are a serologic hallmark of primary biliary cirrhosis (PBC). These react with three structurally and functionally related multienzymic complexes, the 2‐oxoacid dehydrogenase complexes, but chiefly with the E2 subunit of pyruvate dehydrogenase complex (PDC‐E2). Their very close (95%) and specific association with PBC underpins the autoimmune concept of pathogenesis of that disease, notwithstanding several non‐congruent features. Detailed studies, including structural analysis of epitopes, do not disclose how these autoantibodies originate. Their ubiquity in PBC has overshadowed the existence of a second set of relatively PBC‐specific autoantibodies to nuclear antigens for which reactants have been cloned and characterized. These include centromeric proteins; proteins of the nuclear pore complex; nuclear dot proteins, which include Sp‐100 and the promyelocytic leukemia antigen; and a recently identified autoantigen, SOX13. Certain of these reactants are DNA‐binding proteins with transcriptional regulatory activity. Thus serum from individuals with the same clinical syndrome can have autoimmune reactivity to disparate mitochondrial and nuclear constituents in different cellular compartments. Antibody probing of phage displayed random peptide libraries, together with epitope scanning using overlapping sequential octameric peptides from the PDC‐E2 sequence, showed that the discontinuous motifs MH, FV(E) and SYP contributed to a predicted conformational antibody epitope in the inner lipoyl domain of PDC‐E2.

Conformational Epitopes on the Diabetes Autoantigen GAD65 Identified by Peptide Phage Display and Molecular Modeling

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

Direct measurement of cell numbers in microtitre plate cultures using the fluorescent dye SYBR green I

The quantitation of cell numbers is essential for many experimental procedures. This study describes the use of the fluorescent DNA binding dye, SYBR green I, to quantitate accurately cell numbers in the wells of microtitre plates by determination of DNA content. The assay involves a single-step procedure and is sensitive to as few as 1000 cells/well. The advantages of SYBR green I are the low level of background fluorescence in the absence of DNA and the similarities of its absorption and emission spectra to those of fluorescein, which makes it possible to use a wide range of equipment for detection of the amount of bound dye. A particular application of the method is the normalisation of data generated by enzyme-linked immunosorbent assays on whole cells. The procedure is applicable to both fixed and unfixed cells, although for fixed cells permeabilisation provides for greater sensitivity.

Evaluation of ICA512As in Combination With Other Islet Cell Autoantibodies at the Onset of IDDM

OBJECTIVE The ICA512 pancreatic islet autoantigen is a putative tyrosine phosphatase that is co-identified with the earlier described 40-kDa autoantigen. We report the frequency of autoantibodies to islet cell antigen 512 (ICA512As) in recent-onset IDDM and compare this with other islet cell autoantibodies, including those to GAD (GADAs), insulin (IAAs), and islet cell cytoplasm (ICAs) identified by immunofluorescence. RESEARCH DESIGN AND METHODS Sera from 232 children aged between 9 months and 14.9 years collected within 14 days of diagnosis were tested for ICA512As by a radioimmunoprecipitation assay. The results were compared with previously reported data for GADAs (n = 232), IAAs (n = 167), and ICAs (n = 230). RESULTS The frequency of a positive result for ICA512As in children with newly diagnosed IDDM was 60%. The frequency was greater for children with an age of onset between 5 and 10 years (69%) than for children aged < 5 years (49%) and aged between 10 and 15 years (56%). The frequencies for other autoantibody reactivities were 69% for GADAs, 65% for IAAs, and 70% for ICAs. A combination of positive results for ICA512As, GADAs, and IAAs gave a sensitivity for the diagnosis of childhood IDDM of 95%, which was not significantly increased by a positive result for ICAs (96%). CONCLUSIONS Our results further establish that positivity in a combination of tests is more valuable for the prediction of IDDM than a result for any single autoantibody and that the age of the patient should be considered when selecting the combination of tests to use.

Dietary Microbial Toxins and Type 1 Diabetes

Abstract: Toxins may promote type 1 diabetes by modifying or damaging the β cell causing release of autoantigens. Streptomyces is a common soil bacterium that produces many toxic compounds. Some Streptomyces can infect vegetables, raising the possibility of dietary exposure to toxins. We aimed to identify toxins that erode cellular proton gradients in extracts of Streptomyces and infested vegetables and to establish the effect of low doses of these toxins on pancreatic islets in mice. The vacuolar ATPase inhibitors, bafilomycin and concanamycin, and the ionophore, nigericin, were identified in extracts from 4 of 13 Streptomyces isolated from infested potatoes and in potatoes themselves. Injection of bafilomycin A1 into mice impaired glucose tolerance, reduced islet size, and decreased relative β cell mass. Thus, exposure to small quantities of bafilomycin in the diet may contribute to the cause of type 1 diabetes.

Maternal diet during pregnancy and islet autoimmunity in offspring

Background:  Recent studies on the etiology of type 1 diabetes mellitus (T1DM) suggest that the components of the infant diet are associated with islet autoimmunity (IA), a precursor of T1DM. The role of prenatal nutritional exposures has not been thoroughly investigated.

Monoclonal antibody screening of a phage-displayed random peptide library reveals mimotopes of chemokine receptor CCR5: implications for the tertiary structure of the receptor and for an N-terminal binding site for HIV-1 gp120.

The chemokine receptor CCR5 contains seven transmembrane‐spanning domains. It binds chemokines and acts as co‐receptor for macrophage (m)‐tropic (or R5) strains of HIV‐1. Monoclonal antibodies (mAb) to CCR5, 3A9 and 5C7, were used for biopanning a nonapeptide cysteine (C)‐constrained phage‐displayed random peptide library to ascertain contact residues and define tertiary structures of possible epitopes on CCR5. Reactivity of antibodies with phagotopes was established by enzyme‐linked immunosorbent assay (ELISA). mAb 3A9 identified a phagotope C‐HASIYDFGS‐C (3A9 / 1), and 5C7 most frequently identified C‐PHWLRDLRV‐C (5C7 / 1). Corresponding peptides were synthesized. Phagotopes and synthetic peptides reacted in ELISA with corresponding antibodies and synthetic peptides inhibited antibody binding to the phagotopes. Reactivity by immunofluorescence of 3A9 with CCR5 was strongly inhibited by the corresponding peptide. Both mAb 3A9 and 5C7 reacted similarly with phagotopes and the corresponding peptide selected by the alternative mAb. The sequences of peptide inserts of phagotopes could be aligned as mimotopes of the sequence of CCR5. For phage 3A9 / 1, the motif SIYD aligned to residues at the N terminus and FG to residues on the first extracellular loop; for 5C7 / 1, residues at the N terminus, first extracellular loop, and possibly the third extracellular loop could be aligned and so would contribute to the mimotope. The synthetic peptides corresponding to the isolated phagotopes showed a CD4‐dependent reactivity with gp120 of a primary, m‐tropic HIV‐1 isolate. Thus reactivity of antibodies raised to CCR5 against phage‐displayed peptides defined mimotopes that reflect binding sites for these antibodies and reveal a part of the gp120 binding sites on CCR5.

Inhibition of Mitochondrial Complex I May Account for IDDM Induced by Intoxication With the Rodenticide Vacor

Human intoxication with the rodenticide Vacor [N-3-pyridylmethyl-N′-p-nitrophenyl urea or 1-(4-nitrophenyl)-3-(3-pyridylmethyl) urea] induces acute IDDM. We report here that Vacor specifically inhibits the NADH:ubiquinone reductase activity of complex I in mammalian mitochondria. The activity of other respiratory enzymes of mitochondria is unaffected by Vacor at concentrations that completely inhibit the redox and energetic function of complex I. Vacor inhibition of complex I activity quantitatively correlates with the inhibition of insulin release in insulinoma cells and pancreatic islets and is also consistent with the doses reported in cases of human poisoning. These results indicate that the toxic and diabetogenic action of Vacor primarily derives from the inhibition of mitochondrial respiration of NAD-linked substrates in the high-energy demanding cells of the pancreatic islets. This newly identified mechanism of the pathological effects resulting from Vacor intoxication could constitute a paradigm in which to understand environmental or metabolic causes of IDDM.

Antibodies to diabetes-associated autoantigens in Indian patients with Type 1 diabetes: prevalence of anti-ICA512/IA2 and anti-SOX13

We ascertained frequencies of autoantibodies to a suite of islet cell antigens including ICA512/IA2 and SOX13 in Asian Indians with Type 1 diabetes and in other forms of diabetes. Autoantibodies to ICA512/IA2 and SOX13 were tested by radioimmunoprecipitation assay, and results were amalgamated with previous data on antibodies to glutamic acid decarboxylase (GAD) and to islet cell cytoplasmic antigens (ICA). The frequency of anti-SOX13 was higher in Asian Indians than in Europids. Overall, the combined frequency for all autoantibodies to diabetes-associated antigens in Type 1 diabetes in Indians approached the frequency reported for Europids. There was an unexpectedly high frequency of autoantibody reactions to any one of the autoantigens tested (24%) in fibrocalculous pancreatic diabetes, however, individual autoantibody frequencies were relatively low. Our data indicate that, whatever the population studied, testing for multiple autoantigenic reactivities is more informative than more limited testing, and that there may be regional (presumably ethnically based) differences in levels of particular autoantibodies in cases of Type 1 diabetes.