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Biomedical subjects

I R Mackay

Publications and source records attributed to I R Mackay.

At least 19 recordsLinked to original sources
PubMed1992

Mitochondrial DNA mutation associated with aging and degenerative disease.

Previous theories of aging based on somatic mutation neglected mtDNA, which has a high propensity for mutational error. Knowledge of yeast mtDNA mutations and their functional effects, and of human mtDNA mutations identified in the mitochondrial cytopathies, provides for a concept of aging based on the cumulative effect of mutations affecting human mtDNA. An essential feature of this concept is heteroplasmy, representing mixtures of normal and mutant mtDNA at the cellular and mitochondrial level, resulting in a "tissue mosaic" of focal bioenergetic deficits. Direct evidence for the concept is provided by (i) focal loss of staining for mitochondrially encoded enzymes, such as cytochrome c oxidase, in tissues of aged individuals (humans and rats) and (ii) an age-related increase in deletional mutations in mtDNA demonstrable by application of the polymerase chain reaction to DNA templates from individuals of different ages.

Aging↗
PubMed1992

Antiproliferative potencies of interferons on melanoma cell lines and xenografts: higher efficacy of interferon beta.

BACKGROUND: Human melanomas have shown only limited responsiveness to clinical therapy with interferon (IFN). PURPOSE: Our aim was to determine the most effective class of IFN for inhibiting growth of melanoma cells and to establish whether variation exists in response of various cell lines to different IFNs. METHODS: We compared the direct antiproliferative effects of the type I IFN alpha-2b, IFN alpha-4a, and IFN-beta and the type II IFN-gamma on eight melanoma cell lines grown in vitro. We did this comparison by determining the concentration of each IFN that resulted in 50% growth inhibition, using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium tetrazolium bromide] dye uptake method. We also tested IFN alpha-2a and IFN-beta for their ability to inhibit the growth of xenografts of the LiBr melanoma cell line in vivo in nude mice. Receptor binding was determined using [35S]methionine-labeled IFN alpha-4a, in competition with unlabeled IFN alpha-2b, IFN alpha-4a, and IFN-beta. RESULTS: The melanoma cell lines differed markedly in their sensitivity to the IFNs tested: Five were sensitive to low concentrations (less than 30 pM) of IFN-beta, only one was sensitive to similar concentrations of IFN alpha-2b, and none were sensitive to IFN alpha-4a at concentrations up to 920 pM. For all cell lines, the antiproliferative potency of the type I IFNs was IFN-beta greater than IFN alpha-2b greater than IFN alpha-4a. IFN-gamma was less active than IFN-beta on all except one of the cell lines. Similarly, IFN-beta was more potent than IFN alpha-2a in inhibiting the growth of the LiBr xenograft in nude mice. Labeled IFN alpha-4a bound with high specificity in all four melanoma lines tested, and competitive binding experiments showed that the order of binding affinity (IFN-beta greater than IFN alpha-2b greater than IFN alpha-4a) correlated with the order of antiproliferative potency. CONCLUSION: The finding that melanoma cell lines differ intrinsically in their sensitivity to IFNs may explain differences in clinical response. Our results suggest that IFN-beta may be the most effective IFN in the treatment of melanoma, although confirmation will require clinical trials involving large numbers of patients.

Animals↗
PubMed1992

Epitope specificity of antibodies to type II collagen in rheumatoid arthritis and systemic lupus erythematosus.

Antibodies to human type II collagen were examined in the sera of 105 patients with rheumatoid arthritis (RA), 44 patients with systemic lupus erythematosus (SLE) and 11 patients who fulfilled the criteria of both diseases (RA-SLE overlap), using a solid-phase radioimmunoassay (RIA). The frequencies of antibodies to native and denatured human type II collagen were 20% and 27% in RA, 14% and 16% in SLE, and 45% and 36% in RA-SLE overlap. The specificity of the antibodies was further examined by inhibition with native and denatured type II collagen, by immunoblotting on native and denatured type II collagen, and by immunoblotting on cyanogen-bromide derived polypeptides of type II collagen. We could not identify any disease-specific patterns of reactivity. Thus, in the three disease groups the antibody response was polyclonal; there were antibody populations that reacted with native and/or denatured collagen, and epitopes could be assigned to at least three CB peptides, CB10.5, CB11 and CB8.

Antibodies↗
PubMed1992

Antibodies to glutamic acid decarboxylase are associated with HLA-DR genotypes in both Australians and Asians with type 1 (insulin-dependent) diabetes mellitus.

Antibodies to glutamic acid decarboxylase, previously known as the 64 kD antigen, appear to be more predictive of Type 1 (insulin-dependent) diabetes mellitus in Caucasoids than other autoantibodies to islet cell antigens. However, seropositivity to glutamic acid decarboxylase is not universal at the onset of Type 1 diabetes and the prevalence in Asians is low compared to Caucasoid patients. This suggests the involvement of multiple pancreatic autoantigens in the Type 1 diabetes autoimmune process or, genetic differences within and between ethnic groups that contribute to the heterogeneous autoimmune response to glutamic acid decarboxylase or both. Alternatively some cases of Type 1 diabetes could have an aetiology unrelated to autoimmunity. This study examined the differential response to glutamic acid decarboxylase according to HLA-DR and -DQ genotypes, as determined by RFLP, in 49 white Australian and 44 Asian patients with Type 1 diabetes. Among Australians heterozygous for HLA-DR3, DR4, 85% were positive for antibodies to glutamic acid decarboxylase, significantly different (p = 0.039) from the prevalence of 48% in patients with at least one HLA-DR antigen other than DR3 or DR4. Also, among Australians, the presence of "low risk" HLA-DQ antigens, namely DQw5, DQw6 or DQw7, reduced the prevalence of antibodies to glutamic acid decarboxylase by 40% (p = 0.064). Among Asians with Type 1 diabetes and with antibodies to glutamic acid decarboxylase, HLA-DR9 was significantly (p = 0.037) increased in frequency, at 63% compared with 22% in those without glutamic acid decarboxylase antibodies, and the presence of a "low risk" HLA-DQ allele reduced the antibody rates by 87% (p = 0.003).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Differing epitope selection of experimentally-induced and natural antibodies to a disease-specific autoantigen, the E2 subunit of pyruvate dehydrogenase complex (PDC-E2).

Naturally-occurring autoantibodies to a family of mitochondrial enzymes, the 2-oxoacid dehydrogenase complexes (2-OADC), characterize the human liver disease primary biliary cirrhosis. The immunodominant epitope for these autoantibodies is associated with the lipoyl-binding domain of the E2 subunit of the enzymes. The reactivity of these disease-associated autoantibodies was compared with that of antibodies raised in rats and rabbits, by immunization with various preparations derived from the 2-OADC enzymes, using immunization protocols that have successfully induced various organ-specific autoimmune diseases in animals. The immunogens included the intact pyruvate dehydrogenase complex (PDC) from bovine heart, human recombinant PDC-E2, and short synthetic peptides representing the immunodominant lipoic acid binding sequences of the 2-OADC enzymes. The techniques for antibody analysis included immunofluorescence, immunoblotting on mitochondrial extracts, ELISAs using entire PDC, PDC-E2, or synthetic peptides, epitope mapping by peptide scanning on overlapping octameric peptides representing the human PDC-E2 sequence, affinity purification on PDC-E2, and inhibition in vitro by sera of the catalytic function of PDC. Experimental immunization did not elicit any evidence of autoimmune disease. Moreover, the experimentally-induced antibodies in striking contrast to the natural autoantibodies showed preferential reactivity with PDC-E2 rather than with intact PDC, failed to inhibit in vitro the catalytic function of PDC, and, on peptide scanning, reacted with discrete epitopes, but at sites other than the lipoyl-binding region of PDC-E2. Our data indicate that 'multisystem' autoimmune diseases including primary biliary cirrhosis may not be elicitable experimentally because a critical disease-relevant autoepitope is not engaged by the immune system.

Amino Acid Sequence↗
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↗