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L C Harrison

Publications and source records attributed to L C Harrison.

At least 73 records · Page 4Linked to original sources
PubMed1997

Identification of pancreatic beta cell-related genes by representational difference analysis.

A knowledge of beta cell-specific gene expression provides a basis for identifying proteins potentially involved in beta cell function and pathology. To identify candidate beta cell-specific genes, we applied the PCR-based subtractive hybridization technique of representational difference analysis (RDA) to the mouse SV40-transformed endocrine cell lines, betaTC3 and alphaTC1. Following three successive subtractions of alphaTC1 complementary DNA from betaTC3 complementary DNA, difference products were cloned into pUC19 and nucleotide sequences determined. Comparison of 91 sequences against the databases identified 11 known and 8 novel genes. Known genes included previously reported beta cell-specific genes, insulin I/II and islet amyloid polypeptide, as well as other non-beta cell-specific genes such as those for insulin-like growth factor II, selenoprotein P, neuronatin, prohormone convertase, and type 1 protein kinase A regulatory subunit. By Northern blot hybridization, expression of the majority of known and novel genes was restricted to betaTC3 cells. Novel genes BA-12, -13, -14, and -18 were expressed not only in betaTC3 cells, but also in normal pancreatic islets and a limited number of other tissues. The deduced amino acid sequence of BA-14 showed significant homology with members of the cadherin superfamily indicating that BA-14 may encode a cadherin-like molecule potentially involved in beta cell adhesion events during islet ontogeny. In betaTC3 cells, none of the novel genes were regulated at the RNA level by high glucose. However, in parallel studies, transcription of BA-12 was significantly increased by both sodium butyrate and nicotinamide, suggesting that this gene may play a role in pancreatic beta cell growth and/or differentiation. In this study, we have demonstrated that cRDA is an effective strategy for systematically mapping differences in gene expression between two related but functionally-distinct endocrine cells. Its application to experimental animal models of islet-cell regeneration may facilitate the discovery of potential factors that mediate beta cell growth and differentiation.

Amino Acid Sequence↗
PubMed1997

Transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice.

IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell-dependent autoimmune disease in which the beta-cells of the pancreatic islets are destroyed. Several putative beta-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are beta-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.

Aging↗
PubMed1997

Strategies for identifying and predicting islet autoantigen T-cell epitopes in insulin-dependent diabetes mellitus.

T cells recognize peptide epitopes bound to major histocompatibility complex molecules. Human T-cell epitopes have diagnostic and therapeutic applications in autoimmune diseases. However, their accurate definition within an autoantigen by T-cell bioassay, usually proliferation, involves many costly peptides and a large amount of blood. We have therefore developed a strategy to predict T-cell epitopes and applied it to tyrosine phosphatase IA-2, an autoantigen in IDDM, and HLA-DR4(*0401). First, the binding of synthetic overlapping peptides encompassing IA-2 was measured directly to purified DR4. Secondly, a large amount of HLA-DR4 binding data were analysed by alignment using a genetic algorithm and were used to train an artificial neural network to predict the affinity of binding. This bioinformatic prediction method was then validated experimentally and used to predict DR4 binding peptides in IA-2. The binding set encompassed 85% of experimentally determined T-cell epitopes. Both the experimental and bioinformatic methods had high negative predictive values, 92% and 95%, indicating that this strategy of combining experimental results with computer modelling should lead to a significant reduction in the amount of blood and the number of peptides required to define T-cell epitopes in humans.

Autoantigens↗
PubMed1997

Application of genetic search in derivation of matrix models of peptide binding to MHC molecules.

T cells of the vertebrate immune system recognise peptides bound by major histocompatibility complex (MHC) molecules on the surface of host cells. Peptide binding to MHC molecules is necessary for immune recognition, but only a subset of peptides are capable of binding to a particular MHC molecule. Common amino acid patterns (binding motifs) have been observed in sets of peptides that bind to specific MHC molecules. Recently, matrix models for peptide/MHC interaction have been reported. These encode the rules of peptide/ MHC interactions for an individual MHC molecule as a 20 x 9 matrix where the contribution to binding of each amino acid at each position within a 9-mer peptide is quantified. The artificial intelligence techniques of genetic search and machine learning have proved to be very useful in the area of biological sequence analysis. The availability of peptide/MHC binding data can facilitate derivation of binding matrices using machine learning techniques. We performed a simulation study to determine the minimum number of peptide samples required to derive matrices, given the pre-defined accuracy of the matrix model. The matrices were derived using a genetic search. In addition, matrices for peptide binding to the human class I MHC molecules, HLA-B35 and -A24, were derived, validated by independent experimental data and compared to previously-reported matrices. The results indicate that at least 150 peptide samples are required to derive matrices of acceptable accuracy. This result is based on a maximum noise content of 5%, the availability of precise affinity measurements and that acceptable accuracy is determined by an area under the Relative Operating Characteristic curve (Aroc) of > 0.8. More than 600 peptide samples are required to derive matrices of excellent accuracy (Aroc > 0.9). Finally, we derived a human HLA-B27 binding matrix using a genetic search and 404 experimentally-tested peptides, and estimated its accuracy at Aroc > 0.88. The results of this study are expected to be of practical interest to immunologists for efficient identification of peptides as candidates for immunotherapy.

Amino Acid Sequence↗
PubMed1996

Aerosol insulin induces regulatory CD8 gamma delta T cells that prevent murine insulin-dependent diabetes.

Cellular immune hyporesponsiveness can be induced by the presentation of soluble protein antigens to mucosal surfaces. Most studies of mucosa-mediated tolerance have used the oral route of antigen delivery and few have examined autoantigens in natural models of autoimmune disease. Insulin is an autoantigen in humans and nonobese diabetic (NOD) mice with insulin-dependent diabetes mellitus (IDDM). When we administered insulin aerosol to NOD mice after the onset of subclinical disease, pancreatic islet pathology and diabetes incidence were both significantly reduced. Insulin-treated mice had increased circulating antibodies to insulin, absent splenocyte proliferation to the major epitope, insulin B chain amino acids 9-23, which was associated with increased IL-4 and particularly IL-10 secretion, and reduced proliferation to glutamic acid decarboxylase, another islet autoantigen. The ability of splenocytes from insulin-treated mice to suppress the adoptive transfer of diabetes to nondiabetic mice by T cells of diabetic mice was shown to be caused by small numbers of CD8 gamma delta T cells. These findings reveal a novel mechanism for suppressing cell-mediated autoimmune disease. Induction of regulatory CD8 gamma delta T cells by aerosol insulin is a therapeutic strategy with implications for the prevention of human IDDM.

Aerosols↗
PubMed1996

Cytokine regulation of glutamate decarboxylase biosynthesis in isolated rat islets of Langerhans.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease in which cytokines are thought to play an important role in beta-cell destruction and immune regulation. A major target of beta-cell autoimmunity in IDDM is the enzyme glutamate decarboxylase (GAD). We hypothesized that cytokines in the insulitis lesion modulate the synthesis of GAD. This may, in turn, modify the rate of beta-cell destruction. Accordingly we cultured rat islets in the presence and absence of cytokines, and measured synthesis of both isoforms of GAD, GAD65 and GAD67, by [35S]methionine incorporation and immunoprecipitation with a rabbit antiserum that recognizes both GAD65 and GAD67. Incubation of islets with interleukin (IL)-1 beta (1 ng/ml, 24 h), tumour necrosis factor alpha (TNF-alpha; 200 units/ml, 24 h) or interferon gamma (IFN-gamma; 500 units/ml, 72 h) significantly decreased the synthesis of both GAD65 and GAD67, but reduced neither total protein synthesis nor insulin accumulation in the medium or content. Incubation of islets for 24 h in IFN-alpha (1000 units/ml), TNF-beta (50 ng/ml), IL 2 (1000 units/ml), IL-4 (100 ng/ml), IL-6 (10 ng/ml), IL-10 (20 ng/ml), IL-12 (10 ng/ml) or transforming growth factor beta 2 (TGF-beta 2; 5 ng/ml) did not significantly alter GAD65 or GAD67 synthesis. Inhibition of GAD65 and GAD67 protein synthesis by IL-1 beta, TNF-alpha or IFN-gamma was reversed by co-incubation with the nitric oxide synthase inhibitor, NG-monomethyl arginine (NMMA). Expression of both GAD65 and GAD67 mRNA, measured by RNase protection assay, was also decreased by IL-1 beta and completely restored to baseline levels by NMMA. Thus the synthesis of both isoforms of islet GAD is selectively decreased in the presence of IL-1 beta, TNF-alpha or IFN-gamma by a NO-mediated mechanism, probably at the level of cytokine gene transcription. As GAD autoimmunity has been previously shown to have a pathogenic role in an animal model of IDDM, its inhibition by cytokines might limit the immune response, thereby regulating the rate of beta-cell destruction in IDDM.

Amino Acid Sequence↗
PubMed1996

Markers on distal chromosome 2q linked to insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a multigenic autoimmune disease. An IDDM susceptibility gene was mapped to chromosome 2q34. This gene may act early in diabetogenesis, because "preclinical" individuals also showed linkage. Human leukocyte antigen (HLA)-disparate, but not HLA-identical, sibs showed linkage, which was even stronger in families with affected females. The genes encoding insulin-like growth factor-binding proteins 2 and 5 were mapped to a 4-megabase pair interval near this locus. These results indicate the existence of a gene that acts at an early stage in IDDM development, screening for which may identify a specific subset of at-risk individuals.

Alleles↗
PubMed1996

MHCPEP--a database of MHC-binding peptides: update 1995.

MHCPEP is a curated database comprising over 6000 peptide sequences known to bind MHC molecules. Entries are compiled from published reports as well as from direct submissions of experimental data. Each entry contains peptide sequence, MHC specificity and when available, experimental method, observed activity, binding affinity, source protein, anchor positions, as well as publication references. The present format of the database allows text string matching searches but can easily be converted for use in conjunction with sequence analysis packages. The database can be accessed via Internet using Gopher, FTP or WWW.

Computer Communication Networks↗
PubMed1996

The potential roles of endogenous retroviruses in autoimmunity.

Endogenous retroviruses (ERVs) are estimated to comprise up to 1% of human DNA. While the genome of many ERVs is interrupted by termination codons, deletions or frame shift mutations, some ERVs are transcriptionally active and recent studies reveal protein expression or particle formation by human ERVs. ERVs have been implicated as aetiological agents of autoimmune disease, because of their structural and sequence similarities to exogenous retroviruses associated with immune dysregulation and their tissue-specific or differentiation-dependent expression. In fact, retrovirus-like particles distinct from those of known exogenous retroviruses and immune responses to ERV proteins have been observed in autoimmune disease. Quantitatively or structurally aberrant expression of normally cryptic ERVs, induced by environmental or endogenous factors, could initiate autoimmunity through direct or indirect mechanisms. ERVs may lead to immune dysregulation as insertional mutagens or cis-regulatory elements of cellular genes involved in immune function. ERVs may also encode elements like tax in human T-lymphotrophic virus type I (HTLV-I) or tat in human immunodeficiency virus-I (HIV-I) that are capable of transactivating cellular genes. More directly, human ERV gene products themselves may be immunologically active, by analogy with the superantigen activity in the long terminal repeat (LTR) of mouse mammary tumour viruses (MMTV) and the non-specific immunosuppressive activity in mammalian type C retrovirus env protein. Alternatively, increased expression of an ERV protein, or expression of a novel ERV protein not expressed in the thymus during acquisition of immune tolerance, may lead to its perception as a neoantigen. Paraneoplastic syndromes raise the possibility that novel ERV-encoded epitopes expressed by a tumour elicit immunity to cross-reactive epitopes in normal tissues. Recombination events between different but related ERVs, to whose products the host is immunologically tolerant, may also generate new antigenic determinants. Frequently reported humoral immunity to exogenous retrovirus proteins in autoimmune disease could be elicited by cross-reactive ERV proteins. A review of the evidence implicating ERVs in immune dysfunction leads to the conclusion that direct molecular studies are likely to establish a pathogenic role for ERVs in autoimmune disease.

Animals↗
PubMed1996

Both TH1 and TH2 cytokine mRNAs are expressed in the NOD mouse pancreas in vivo.

In NOD mice, autoimmune recognition and destruction of pancreatic islet beta-cells appear to be independently regulated: all mice develop cellular infiltration of the islets (insulitis), but not all develop diabetes. The destructive potential of the insulitis lesion may depend on the balance between the two CD4+ T-cell subsets. TH1 and TH2, that mediate cellular-cytotoxic and humoral responses, respectively. With a semi-quantitative reverse transcriptase-PCR assay, we examined whether the disease process was reflected in the profiles of TH1 (IL-2, IFN-gamma and IL-12) and TH2 (IL-4, IL-6 and IL-10) cytokine mRNAs expressed in pancreata of NOD mice. Pancreata rather than isolated islets were examined to minimize manipulation ex vivo to preserve the expression of cytokine transcripts in vivo. At age 6 weeks, when 70% of mice had insulitis, all cytokine transcripts were detected in most pancreata, and their expression levels corresponded to the degree of insulitis. Similarly, during induction of diabetes with cyclophosphamide all transcripts were detected and levels corresponded with the degree of insulitis. In one-year-old mice without diabetes, all transcripts were detected but levels did not correspond to the degree of insulitis. Thus, in pancreata of NOD mice with different degrees of insulitis, we were unable to demonstrate, at the RNA level, polarisation of cytokine expression into either a TH1 or TH2 profile. This finding does not, however, exclude expression of distinct cytokine transcripts by immuno-inflammatory cells within the islet lesion, which might be revealed by in situ hybridization.

Aging↗
PubMed1995

Cytokine-based human whole blood assay for the detection of antigen-reactive T cells.

The measurement of cytokines produced by activated T cells refines assessment of cellular immune function and facilitates whole blood T cell assays. The latter approximate conditions in vivo and obviate the need to purify blood mononuclear cells. We have investigated the parameters of the whole blood assay in humans to standardize and optimize the detection of tetanus-specific T cell cytokine responses. Optimal conditions include the use of undiluted whole blood, an incubation time of 36-48 h and a minimum of delay between venesection and incubation of the blood with antigen. Blood should be drawn at a standard time of day to minimize inter-assay variation due to diurnal rhythmicity in cytokine production. Interferon-gamma or interleukin-2 are specific and reliable readouts; other cytokines can be measured to further characterize the TH1 and TH2 elements of the T cell responses, although tetanus-stimulated IL-4 production is detected in only a minority of healthy individuals. The whole blood assay is a potentially valuable tool for assessing cellular immune function and screening for antigen-reactive T cells in humans.

Blood Preservation↗
PubMed1995

Transcription and translation of two glutamate decarboxylase genes in the ileum of rat, mouse and guinea pig.

gamma-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter, synthesised from glutamate by glutamate decarboxylase (GAD), in the central nervous system. Two forms of GAD, designated GAD 65 and GAD 67, are encoded by distinct genes and have been demonstrated in the mammalian brain. GABA has been postulated to be synthesised in neurons of the enteric nervous system (ENS), but evidence for its role as an enteric neurotransmitter is equivocal. We therefore aimed to determine whether GAD 65 and GAD 67 messenger RNAs (mRNAs) and proteins were expressed in the ileum of mice, rats and guinea pigs. Using an RNase protection assay, both GAD 65 and GAD 67 mRNAs were detected in the rodent small intestine. Antisera specific for GAD 65 or GAD 67, used in immunoblot analyses, revealed GAD 65-like and GAD 67-like immunoreactivity in rat and guinea pig ileum. Anti-GAD 65 antisera detected a major band of 65 kDa. Anti-GAD 67 antisera detected a major band of 55 kDa, which probably represented a breakdown product, and a minor band of 67 kDa. Analysis of immunoblot extracts of rat and guinea pig ileum revealed more GAD 67-like than GAD 65-like immunoreactivity. GAD enzymatic activity was high in the rat and guinea-pig brain, and low in the whole and dissected ileum. These results demonstrate that both GAD 65 and GAD 67 genes are transcribed and translated in the ileum of three rodent species and lend indirect support to the postulate that GABA is synthesised by neurons of the ENS and intestinal endocrine cells.

Amino Acid Sequence↗
PubMed1995

Antibodies to synovial antigens in recent-onset rheumatoid arthritis.

OBJECTIVE: To identify synovial antigens that bind to serum antibodies from subjects with recent-onset rheumatoid arthritis (RA) (< 6 months of synovitis). METHODS: Soluble and insoluble fractions of Triton X-100 extracts of RA and normal synovial tissue, and normal spleen and placenta, were immunoblotted with sera from 27 patients with recent-onset RA, 13 autoimmune disease control subjects, and 13 blood bank control donors. Bound immunoglobulin was probed with 125I-labeled protein A. RESULTS: Antibodies in the sera of 20 (74%) patients with recent-onset RA recognized at least 1 of 5 antigens in both a disease- and tissue-specific and nonspecific manner. Anti-La antibodies, usually associated with primary Sjögren's syndrome, were detected in 2 sera. Eight sera had increased reactivity to an IgG heavy and light chain dimer. There was strong binding of 8 sera with a 35-kd doublet and of 3 sera with a 55-kd species in RA and normal synovial lysates (insoluble fractions). Two sera uniquely recognized a 45-kd protein only in the RA synovial lysate (soluble fraction). CONCLUSION: IgG antibodies in the sera of patients with recent-onset RA show positive immunoblots for 3 novel synovial antigens of 35-kd, 55-kd, and 45-kd, as well as for 2 previously characterized antigens (La and IgG). Thus, a variety of synovial antigens appear to be recognized by B cells even early in the clinical course of RA.

Adolescent↗
PubMed1995

HLA antigens and age at diagnosis of insulin-dependent diabetes mellitus.

IDDM results from the immune-mediated destruction of pancreatic islet beta cells. Clinicopathologic heterogeneity in IDDM is reflected in part by the wide age range over which the onset of clinical symptoms can occur, after months to years of subclinical "insulitis." Because MHC genes play a critical role in immune function we studied their possible contribution to IDDM heterogeneity by analyzing HLA profiles of 194 IDDM patients in relation to their age at diagnosis. Restriction of HLA-DR heterogeneity was observed in patients diagnosed before age 21 years. Frequencies of DR3 and DR3/4 were highest in the < or = 6-year-old age group and thereafter declined with increasing age at diagnosis. In contrast, the frequency of DR4 remained increased up to age 30 years at diagnosis. DR7, normally considered to be a neutral allele, was like DR2 and DR5, significantly decreased in patients diagnosed before age 21 years. The A30-B18-DR3 haplotype was significantly increased in the < or = 6-year-old age group, A1-B8-DR3 was increased in the > or = 31-year-old group. B62-DR4 was increased only in the > 12-year-old age group. In DR4 patients the frequency of DQ8 was increased across all age groups. A sex difference was observed in those diagnosed at < or = 12 years of age, with an excess of females in the DR3+/DR4- group and males in the DR3-/DR4+ group. An association of DPB1 with IDDM was revealed by an increased frequency overall of DPB1*0301 and/or DPB1*0401, being more pronounced in patients diagnosed at > 20 years of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗