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Biomedical subjects

I R Mackay

Publications and source records attributed to I R Mackay.

At least 127 records · Page 7Linked to original sources
PubMed1992

Prevention of spontaneous and cyclophosphamide-induced diabetes in non-obese diabetic (NOD) mice with oral 2-acetyl-4-tetrahydroxybutylimidazole (THI), a component of caramel colouring III.

The effect of oral administration of THI, a compound present in ammonia caramel food colouring, was studied in spontaneous and induced murine diabetes mellitus. Continuous administration of THI at 400 ppm in drinking water reduced the prevalence of spontaneous diabetes in female NOD/Lt mice from 63% in untreated controls to 8% in treated animals. Since cyclophosphamide (CP) accelerates and intensifies diabetes in NOD mice, we also studied the effect of THI in this model. Diabetes incidence was reduced from 100% in mice given only CP to 13-14% in mice given THI either concurrently or from 14 days previously. Histologically, THI greatly reduced the severity of insulitis. As measured by flow cytometry, all THI-treated mice had a 60-80% reduction in splenic CD4+ and CD8+ T cells. THI-treated mice showed no untoward effects and specifically no weight loss, or pathological changes in their livers, kidneys or lungs. However, there was moderate atrophy of the thymus cortex. THI is a small imidazole-containing compound with structural similarity to histamine and urocanic acid, both known to have immunosuppressive properties. It is a widely used food additive with no known long-term toxic effects at low dosage. Thus, THI could be a useful immunosuppressive agent.

Administration, Oral↗
PubMed1992

Comparative immunoreactive profiles of Japanese and American patients with primary biliary cirrhosis against mitochondrial autoantigens.

Primary biliary cirrhosis (PBC) has been described among various ethnic and racial populations in all parts of the world. However, the incidence and prevalence of PBC varies considerably in different geographic areas. It has the highest frequency in Northern Europe, is considerably lower in Japan and still lower in other parts of Asia. There has not hitherto been a detailed immunological profile of antimitochondrial antibodies according to geographic region. We have used recombinant or purified preparations from the 2-oxo-acid dehydrogenase enzyme complexes, the major mitochondrial autoantigens in PBC (PDC-E2, BCOADC-E2, OGDC, protein X and PDC-E1 alpha) to compare the reactivity of sera from either similarly staged sera from Japanese (n = 23) or American-Caucasian patients (n = 39) with PBC. In all cases, the first available sera following diagnosis was selected. Interestingly, only 65% of Japanese patients reacted by ELISA with PDC-E2 compared with more than 95% of the North American group. Moreover, the level of enzyme-inhibitory antibodies to PDC was lower in the Japanese. Our findings prompt the need for characterization of specific susceptibility genes and environmental factors in various parts of the world to clarify the etiology of PBC.

Antigen-Antibody Reactions↗
PubMed1992

Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-M(r) islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3-42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means +/- 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.

Adolescent↗
PubMed1992

Molecular biology of the 2-oxo-acid dehydrogenase complexes and anti-mitochondrial antibodies.

The confluence of molecular biology and clinical medicine has provided new and valuable insights in PBC. Our understanding of the immunobiology of PBC has changed dramatically using this new technology. It is now possible to explicitly define mitochondrial autoantigens and examine recognition sites, by using autoantibodies, at the primary sequence level. In addition, cloned antigens have been developed to reliably assay for presence of autoantibodies; the use of cloned recombinant antigens should replace that of traditional immunofluorescence for AMA assay. It is also now possible to begin the task of defining the role of T cells in the immunopathology of PBC and exploring the issue of whether immunotherapy is possible. Finally, there is increasing evidence that PDC-E2 is located on the cell membrane of biliary epithelial cells. The mechanism for this expression remains to be studied. The explosion of data in PBC is an example of the serendipity and synergy brought about by application of new techniques to investigate old problems.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1992

Validation of the MTT dye assay for enumeration of cells in proliferative and antiproliferative assays.

Increasing use is being made of colorimetric assays to quantitate viable cells, e.g., the cellular reduction of the tetrazolium salt, MTT, to formazan by mitochondrial succinate dehydrogenase. We validated this assay for cell proliferation in mixed lymphocyte cultures as compared with 3H-thymidine uptake, and for inhibition of cell proliferation induced by interferon with results compared by direct cell counting. We also found that cells do not, as previously assumed, require functional mitochondria: there were no differences in formazan production by normal cells or respiratory defective cells in which mitochondria had been poisoned by the nucleic acid toxin, ethidium bromide. The high reproducibility of MTT metabolism by lymphoblasts and various cultured cell lines establishes the reliability and versatility of this method for quantitating cell numbers.

Animals↗
PubMed1991

The human pyruvate dehydrogenase complex: a polymorphic region of the lipoate acetyl transferase (E2) subunit gene.

A major issue in the study of the pathogenesis of primary biliary cirrhosis is whether the E2 subunit of the pyruvate dehydrogenase complex (PDH-E2), the major autoantigen in the disease, exists as a tissue-specific isoform. cDNA clones spanning a segment of the 3'-catalytic region of PDH-E2 (nt 1158-1361) have been isolated from human kidney, placenta and bile epithelium cells. Nucleotide sequence analysis of the clones showed differences consistent with the presence of normal variants of PDH-E2 in the human population. However, the existence of tissue-specific isoforms of PDH-E2 cannot yet be discounted.

Acetyltransferases↗
PubMed1991

Oncofetal expression of the human intestinal mucin glycoprotein antigens in gastrointestinal epithelium defined by monoclonal antibodies.

A mucin preparation from a colonic adenocarcinoma was used to prepare monoclonal antibodies (MAbs) that reacted specifically either with normal adult small-intestine mucin antigen(s) (SIMA), or normal adult large-intestine mucin antigen(s) (LIMA). Both SIMA and LIMA show a unique oncofetal pattern of expression. Thus SIMA was expressed in early fetal stomach, large and small intestines but thereafter only in the normal small intestine. SIMA expression was detected immunohistochemically in cancers of the colorectum (82/112) and stomach (48/86). LIMA was detected in the stomach of the early fetus but thereafter only in the normal large intestine. LIMA expression was detected in 61/86 cancers of the stomach. Moreover, both SIMA and LIMA were expressed inappropriately in mucosa adjacent to tumors, indicative of the detection of possible pre-malignant epithelium. We used a sandwich ELISA and biochemical procedures to show that the SIMA and LIMA molecules were large extensively glycosylated multi-unit mucin glycoproteins that differed markedly from each other. SIMA, whether extracted from normal small-intestine or colonic cancers, had a molecular weight above 1.000 kDa, a mean buoyant density 1.33 g/ml and s value of 4.8. LIMA had a molecular weight above 10.000 kDa, a mean buoyant density 1.45 g/ml and an s value 9.5. The SIMA and LIMA epitopes were judged to be carbohydrate in nature by reason of their resistance to harsh physical chemical treatments or protease digestion, and sensitivity to periodate oxidation, neuraminidase or beta elimination. Only the SIMA epitope was sensitive to neuraminidase. In conclusion, MAbs to carbohydrate-dependent epitopes on SIMA and LIMA identify the oncofetal pattern of expression of these distinct intestinal mucin glycoproteins in colonic and gastric carcinoma. These MAbs will be useful in further studies of the significance of oncofetal mucin expression during carcinogenesis.

Adenocarcinoma, Mucinous↗
PubMed1991

Islet cell antibodies and other markers of autoimmunity and diabetes mellitus in Nauruans.

Among the population of Nauru there is a high prevalence of diabetes with onset in early adult life. To ascertain whether autoimmunity to islet cell antigens contributes to this diabetes, a population survey of serum autoantibodies was performed. Subjects were grouped into euglycaemic control subjects, those with impaired glucose tolerance, and those with diabetes subdivided according to duration of disease. No subject was positive by immunofluorescence for islet cell autoantibodies. Various other autoantibodies to nuclear, thyroid and gastric autoantigens were detectable, at comparable frequencies in the three groups. This population study on Nauruan subjects selected to include those in the early phases of disease negates a contribution from islet cell autoimmunity, and thus supports the concept that the disease is the Type 2 (non-insulin-dependent) type.

Animals↗
PubMed1991

Primary biliary cirrhosis: paradigm or paradox for autoimmunity.

Primary biliary cirrhosis has been classified as a model autoimmune disease based on striking defects in immune regulation and the presence of autoantibodies to mitochondria. Until recently the significance and definition of mitochondrial autoreactivity was unknown. Since 1987, there has been a vast improvement in the understanding and definition of the biochemical and molecular target autoantigens. The cloning of complementary DNAs for mitochondrial antigens has led to the identification of three enzymes of the 2-oxo-acid dehydrogenase family as the targets of the autoantibodies to mitochondria in patients with primary biliary cirrhosis. The major reactive autoantigen is the E2 subunit of pyruvate dehydrogenase. Immunodominant sites on pyruvate dehydrogenase E2 (autoepitopes) have been mapped and have been shown to be the site of attachment of the functionally important lipoic acid prosthetic group. The autoepitope for the other enzymes probably occupies an equivalent site on the enzyme. The availability and definition of these mitochondrial autoepitopes have allowed specific questions to be addressed relating to the processing and targeting of these autoantigens as well as further studies on mechanisms of immunopathology. Similarly, the availability of well-defined autoantigens could contribute to the development of valid animal models in addition to the already described reproduction of the biliary ductular lesions by transfer of peripheral blood lymphocytes from patients with primary biliary cirrhosis into severe combined immunodeficient mice. Such models will facilitate specific study of the role of major histocompatibility complex expression and the characterization of T-cell reactivity. Thus, primary biliary cirrhosis is a key example of significant progress in autoimmunity being made by use of recombinant DNA technology.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1991

Inhibitory autoantibody to a conformational epitope of the pyruvate dehydrogenase complex, the major autoantigen in primary biliary cirrhosis.

The mitochondrial autoantibodies present in primary biliary cirrhosis (PBC) react with the 2-oxoacid dehydrogenase enzymes that include the pyruvate dehydrogenase complex (PDC). All epitopes so far demonstrable, including the inner lipoyl domain of PDC-E2, have been revealed by immunoblotting. To identify other epitopes, advantage was taken of the capacity of PBC sera to inhibit in vitro the catalytic function of the PDC enzyme. PBC sera were analyzed by affinity chromatography, using columns containing either recombinant PDC-E2 or intact PDC. Fractions that bound to the column (B) and nonbinding effluent fractions (NB) were tested by immunoblotting and ELISA and for their capacity to inhibit enzyme function. After separation on the PDC-E2 column the B fractions were reactive with PDC-E2 and intact PDC, whereas the NB fractions did not react by immunoblotting or ELISA with PDC-E2 but did react strongly by ELISA with PDC and did strongly inhibit the enzyme function. After separation of sera on the PDC column, the B fractions reacted more strongly with PDC than PDC-E2 by ELISA and strongly inhibited the enzyme function, whereas the NB fractions were nonreactive. Thus we describe a hitherto undetected population of autoantibodies in PBC sera that react only with intact PDC but not with the recombinant PDC-E2 subunit that contains the lipoyl epitope, are demonstrable by ELISA but not by immunoblotting, and notably, inhibit enzyme function. These nonblotting inhibitory autoantibodies in PBC are presumed to react with an exclusively conformational determinant perhaps presented by the tertiary structure of the entire enzyme complex.

Autoantibodies↗
PubMed1991

Intermittent interferonemia and interferon responses in multiple sclerosis.

In view of the immunoregulatory and antiviral properties of the interferons (IFNs), the production of and response to these cytokines in vivo and in vitro were assessed in 42 patients with multiple sclerosis (MS), a disease with features of autoimmunity and a viral infection. Serum IFN, determined by bioassay of antiviral activity at 10 intervals over 18 months, was detectable at levels ranging from 16 to 250 IU/ml, at least once and up to five times in 37 of the 42 patients. Of 420 samples tested, 88 (21%) were positive. None of the 71 serum samples from 37 healthy subjects contained detectable IFN activity. Neutralization of antiviral activity by antibodies showed that the serum IFN type was IFN-alpha in 82 samples, IFN-gamma only in 2, and both IFN-alpha and IFN-gamma were present in 4. At the initial time point the activity of 2'-5' oligoadenylate synthetase (OAS), an IFN-induced enzyme, was elevated in peripheral blood leucocytes (PBL) from 13 patients, but not in 7 patients seropositive for IFN, indicating that in some patients there was a failure of PBL to respond to endogenous IFN. In most patients the capacity of PBL in vitro to produce IFNs-alpha/beta or -gamma after induction by virus or mitogens, respectively, was likewise reduced. These various abnormalities in IFN responses could not be correlated with clinical assessments of disease activity but may reflect subclinical attacks. The abnormalities described, in particular the intermittent interferonemia in MS, are more striking than in other diseases previously reported, indicating an unusual component to the stimulus for IFN production (viral or other) or the response to it. The effects of endogenous IFN production may have implications for the scheduling of therapy with IFN in MS.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

The identification and cloning of the murine genes encoding the liver specific F alloantigens.

The liver specific F alloantigen is a highly conserved abundant protein found in hepatic cytoplasm; smaller amounts are detected in renal tubule cells and the perikaryon cells of the central nervous system. Although the biological function of the F alloantigen is unknown, the immune response to F has been extensively studied as a murine model of tolerance and autoimmunity. Murine F exists in two allelic forms, designated F type 1 and type 2, each of approximately 43 kDa. The immune response to the allotypic form is restricted to mouse strains of I-Ak. Responding strains immunized with allotypic F break tolerance and produce precipitating antibody that reacts with both allelic forms, i.e., immunogen and self. Thus an autoantibody is produced. Using the previously isolated rat F cDNA as a probe, we report the cloning and sequencing of the two murine F allotypes. These two alleles are nearly homologous except at the extremes of the coding sequence. There are a number of regions within the F sequence that are similar to peptides that interact specifically with I-Ak. In particular, there is a sequence near the carboxy terminus, where the two allotypes differ, that has homology to the I-Ak restricted malarial antigen peptide of the ring-infected erythrocyte surface antigen (RESA).

Amino Acid Sequence↗
PubMed1991

Acute theophylline intoxication.

We describe a case of intentional acute theophylline intoxication with cardiac, cerebral, and gastrointestinal features of moderate-to-severe toxicity. The unusual metabolic and hematologic sequelae included hypokalemia, hyperglycemia, metabolic acidosis, extreme neutrophilia, increased creatinine levels attributed to muscle damage, and hematuria. The implications of these unusual findings for the management of acute theophylline intoxication include the recognition that these effects can be due to intoxication per se and do not necessarily indicate a primary endocrine disorder or infection.

Acidosis↗
PubMed1991

An automated microassay for enzyme inhibitory effects of M2 antibodies in primary biliary cirrhosis.

In primary biliary cirrhosis (PBC), autoantibodies are produced to the M2 group of mitochondrial antigens, of which a major constituent is the E2 subunit of the pyruvate dehydrogenase complex (PDC). These antibodies, in addition to conventional reactivities with PDC, characteristically inhibit the catalytic function of PDC in vitro, as judged by a macroinhibition assay based on spectrophotometry. We describe a microinhibition assay adapted for microtitre plates and for an automated readout of results by an ELISA plate reader. We show that this microassay has a sensitivity similar to that of the macroassay. In a study of 83 sera, from PBC and other diseases, the inhibitory microassay proved specific for PBC. This automated inhibitory microassay for PBC-sera could become a primary laboratory procedure for the diagnosis of PBC, particularly because it may identify antibodies to the actual autoepitope on the PDC-E2.

Animals↗
PubMed1991

Autoantibody against dihydrolipoamide dehydrogenase, the E3 subunit of the 2-oxoacid dehydrogenase complexes: significance for primary biliary cirrhosis.

Autoantibodies in primary biliary cirrhosis recognize mitochondrial 2-oxacid dehydrogenase complexes, particularly the E2 subunits. Reactivity with the E3 subunit, common to each of the enzyme complexes, was sought by immunoblotting, with sera screened at 1:100 instead of the conventional 1:1,000 dilution. This was found in 11 of 29 sera from patients with primary biliary cirrhosis but also in 10 of 40 sera from normal subjects. Two-dimensional immunoblotting and immunoblotting on purified enzymes established that the reactivity was actually with E3 rather than with another component of the 2-oxoacid enzymes of similar molecular weight. Purified antibodies to E3 eluted from an affinity column did not cross-react with other components of the 2-oxoacid enzyme complexes. The antibodies to E3 did not react with the Escherichia coli or yeast E3 subunits, suggesting that they are not stimulated by immune responses against microorganisms. Thus the proposal that reactivity to the shared E3 subunit of the 2-oxoacid enzyme complexes could initiate primary biliary cirrhosis is not reflected at the antibody level.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗