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M A Atkinson

Publications and source records attributed to M A Atkinson.

At least 55 records · Page 3Linked to original sources
PubMed1997

Retardation or acceleration of diabetes in NOD/Lt mice mediated by intrathymic administration of candidate beta-cell antigens.

A single injection of syngeneic islet cells into the thymus of 4-week-old NOD/Lt female mice strongly retards diabetogenesis. The present study used the intrathymic route of antigen administration to compare the relative efficacy of peptides/proteins derived from two major candidate pancreatic beta-cell autoantigens, insulin and GAD65, to modulate diabetogenesis. Intrathymic administration of insulin B chain or recombinant human GAD65 significantly suppressed diabetogenesis during a 20-week follow-up period, whereas no protection was mediated by either insulin A chain or a synthetic peptide (A2) derived from it. Quite unexpectedly, two GAD65-derived peptides near the COOH-terminus (p34 and p35) accelerated diabetes onset. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed on cDNAs from isolated islets or whole pancreases of NOD/Lt females 4 weeks after intrathymic injections. Protection mediated by intrathymic administration with either intact islet cells or GAD65 were correlated with an upregulation of mRNA for T-helper 2 (Th2)-associated cytokines (interleukin [IL]-4, IL-10), concomitant with downregulation of Th1-associated interferon (IFN) transcripts (all normalized to T-cell receptor Cbeta transcripts) in islet-infiltrating lymphocytes. Protection mediated by the intrathymic administration of insulin B chain, however, correlated only with a modest upregulation of IL-4 and IL-10 transcript levels, and no diminution in IFN-gamma transcripts. In contrast, the diabetes-accelerating GAD65 p34 and p35 peptides were not associated with an immune deviation, expressing levels of IFN-gamma characteristic of islet-infiltrating lymphocytes in vehicle-injected NOD controls. Hence, Th1-to-Th2 immune deviation provides only a partial explanation for peptide immunotherapy of diabetes in NOD mice. The finding that certain peptides can accelerate rather than retard diabetogenesis as a function of route and age of administration adds a cautionary note to this type of therapy.

Adoptive Transfer↗
PubMed1997

Molecular mimicry and the pathogenesis of insulin-dependent diabetes mellitus: still just an attractive hypothesis.

An abundant body of literature suggests that the cellular immune system plays a key role in the autoimmune destruction of insulin-secreting pancreatic beta cells that results in insulin-dependent diabetes mellitus (IDDM). For years, studies have supported the concept that molecular mimicry, a process of antigenic crossreactivity resulting from similarity in amino acid sequence or structure, could be one pathway whereby this disease is induced or its natural history modulated. However, the transfer of this 'hypothesis' to that of a proven mechanism underlying this clinical disorder has been slow and never fully achieved. This article reviews the theoretical basis for molecular mimicry in autoimmune disease and the evidence supporting its role in the pathogenesis of IDDM.

Amino Acid Sequence↗
PubMed1996

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF-RecO-RecR complex in DNA repair and recombination.

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF-RecO-RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF-RecO-Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF-RecO-RecR complex functions as an anti-Ssb factor.

Bacterial Proteins↗
PubMed1996

Quantitative thresholds of MHC class II I-E expressed on hemopoietically derived antigen-presenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection.

Two homozygous lines of transgenic NOD/Lt mice expressing MHC class II I-E molecules at quantitatively different levels were utilized to study mechanisms of I-E-mediated diabetes prevention. In line 12, I-E expression on APC at levels comparable with that in BALB/cByJ controls conferred only partial diabetes resistance. In line 5, greater than normal I-E levels on APC correlated with nearly complete resistance. Levels of endogenously encoded I-Ag7 correlated inversely with transgene-induced I-E expression. T cell transfer experiments into NOD/severe combined immunodeficient mice demonstrated the presence of pathogenic T cells in I-E+ donors, and that continuous expression of I-E on hemopoietically derived APC was required to block their pathogenic function. T cells from transgenic and nontransgenic NOD/Lt mice primed in vivo against the beta cell autoantigen 65-kDa isoform of glutamic acid decarboxylase (GAD65) and two peptides derived from this protein proliferated when restimulated in vitro. However, reverse-transcription PCR and ELISA measurements of cytokine mRNA and protein levels showed that the GAD65-reactive T cells from both line 5 and line 12 mice produced higher levels of IL-4 and lower levels of IFN-gamma than similar T cells from standard NOD/Lt mice. Thus, the inverse relationship between I-E and I-Ag7 expression was associated with qualitative differences in T cell responses to putative beta cell autoantigens. Collectively, these data indicate quantitative increases in I-E expression on APC may block insulin-dependent diabetes mellitus by altering the balance of cytokines produced by beta cell autoreactive T cells.

Amino Acid Sequence↗
PubMed1996

Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes.

We previously demonstrated that a spontaneous Th1 response against glutamate decarboxylase (GAD65) arises in NOD mice at four weeks in age and subsequently T cell autoimmunity spreads both intramolecularly and intermolecularly. Induction of passive tolerance to GAD65, through inactivation of reactive T cells before the onset of autoimmunity, prevented determinant spreading and the development of insulin-dependent diabetes mellitus (IDDM). Here, we examined whether an alternative strategy, designed to induce active tolerance via the engagement of Th2 immune responses to GAD65, before the spontaneous onset of autoimmunity, could inhibit the cascade of Th1 responses that lead to IDDM. We observed that a single intranasal administration of GAD65 peptides to 2-3-wk-old NOD mice induced high levels of IgG1 antibodies to GAD65. GAD65 peptide treated mice displayed greatly reduced IFN gamma responses and increased IL-5 responses to GAD65, confirming the diversion of the spontaneous GAD65 Th1 response toward a Th2 phenotype. Consistent with the induction of an active tolerance mechanism, splenic CD4+ (but not CD8+) T cells from GAD65 peptide-treated mice, inhibited the adoptive transfer of IDDM to NOD-scid/scid mice. This active mechanism not only inhibited the development of proliferative T cell responses to GAD65, it also limited the expansion of autoreactive T cell responses to other beta cell antigens (i.e., determinant spreading). Finally, GAD65 peptide treatment reduced insulitis and long-term IDDM incidence. Collectively, these data suggest that the nasal administration of GAD65 peptides induces a Th2 cell response that inhibits the spontaneous development of autoreactive Th1 responses and the progression of beta cell autoimmunity in NOD mice.

Administration, Intranasal↗
PubMed1996

Stiparin: a glycoprotein from sea cucumber dermis that aggregates collagen fibrils.

The interactions between collagen fibrils in many echinoderm connective tissues are rapidly altered by the secretions of resident neurosecretory cells. Recent evidence has suggested that a secreted protein is responsible for the interactions that lead to an increase in tissue stiffness (Trotter and Koob, 1995). Structurally intact collagen fibrils have been isolated from such a connective tissue- the dermis of the sea cucumber Cucumaria frondosa- and used in an assay in vitro to identify a protein that binds to them and causes them to aggregate. This protein has been purified by anion-exchange and molecular sieve chromatography. It is eluted from a MonoQ column at approximately 0.55 M NaCl. Its isoelectric point is 5.2. It elutes from a Superose-6 column in a position corresponding to a molecule with a Stokes radius of 11.5 nm. Its native molecular weight estimated from sedimentation equilibrium analysis under non-denaturing conditions is 375,000, and its monomer molecular weight, estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, is approximately 350,000. Sedimentation velocity measurements indicated for the native molecule a sedimentation coefficient of 11 x 10(-13)s, a diffusion coefficient of 3.274 x 10(-7) cm2s-1, and a frictional ratio of 1.95, which corresponds to a prolate ellipsoid of revolution with an axial ratio of 19. The highly asymmetric structure suggested by the above correlated well with the images obtained by transmission electron microscopy following rotary shadowing, which revealed a flexible structure approximately 125 nm long. Based on its ability to aggregate collagen fibrils, this protein has been named "stiparin," from the Latin stipare, "to pack together."

Animals↗
PubMed1996

Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice.

In nonobese diabetic (NOD) mice, beta-cell reactive T-helper type 1 (Th1) responses develop spontaneously and gradually spread, creating a cascade of responses that ultimately destroys the beta-cells. The diversity of the autoreactive T-cell repertoire creates a major obstacle to the development of therapeutics. We show that even in the presence of established Th1 responses, it is possible to induce autoantigen-specific anti-inflammatory Th2 responses. Immune deviation of T-cell responses to the beta-cell autoantigen glutamate decarboxylase (GAD65), induced an active form of self-tolerance that was associated with an inhibition of disease progression in prediabetic mice and prolonged survival of syngeneic islet grafts in diabetic NOD mice. Thus, modulation of autoantigen-specific Th1/Th2 balances may provide a minimally invasive means of downregulating established pathogenic autoimmune responses.

Adoptive Transfer↗
PubMed1996

Immunological and metabolic effects of prophylactic insulin therapy in the NOD-scid/scid adoptive transfer model of IDDM.

Prophylactic insulin therapy prevents IDDM in spontaneous animal models of the disease and has shown promise in preventing the disease in humans. Although large clinical trials have been formed to use this therapy, a comparative analysis of the efficiency of different pharmaceutical forms and doses of insulin in preventing IDDM has not been performed, and the mechanism underlying the observed prevention of disease is unknown. In the NOD-scid/scid adoptive transfer model of IDDM (10(7) new-onset NOD splenocytes injected intravenously into 6- to 8-week NOD/scid-scid recipients; insulitis develops at 6-9 days post-transfer and 100% IDDM by 32 days post-transfer), life-table (log-rank) analyses revealed that IDDM can be delayed (compared with insulin-free diluent, once daily, n = 8) with equivalent efficiency by prophylactic administration (-9-50 days post-transfer) of high (metabolism-altering) doses of short-acting (0.5 U, once daily, regular, n = 13) or long-acting (0.5 U, once daily, ultralente, n = 9) insulin as well as non-metabolism-altering low-dose insulin (0.02 U, once daily, regular, n = 8). Furthermore, IDDM was delayed with somatostatin (0.2 microgram, twice daily, n = 11), an agent that suppresses endogenous insulin production. No significant difference was seen between the preventative effects of these agents. In an assessment of when therapies can be initiated and still maintain clinical efficiency, only prophylactic somatostatin therapy delayed IDDM (n = 10, P = 0.02) when initiated at 14 days post-transfer, whereas the short-acting insulin regimen did not retard the onset of IDDM (n = 8, P = 0.25) compared with diluent-treated controls. The 24-h urinary C-peptide levels were significantly reduced with short-acting (-56%, P = 0.01) and long-acting (-67%, P = 0.02) insulin products and somatostatin (-59%, P = 0.02) compared with diluent-treated controls. These results indicate that both immunological and metabolic (i.e., beta-cell rest) factors may contribute to the beneficial effects of prophylactic insulin therapy.

Animals↗
PubMed1996

Pharmacokinetics of aminoguanidine administration and effects on the diabetes frequency in nonobese diabetic mice.

In vitro studies suggest that intra-islet nitric oxide production may contribute to the pathogenesis of autoimmune insulin-dependent diabetes mellitus. We tested whether aminoguanidine (AG), a competitive inhibitor of inducible nitric oxide synthase, might block beta cell destruction and prevent insulin-dependent diabetes mellitus in vivo. A total of 50 female nonobese diabetic mice, from the time of weaning until 32 wk of age, received injections (i.p.) twice daily with 50 mg AG/kg body weight and received AG in drinking water (350 mg/liter). A total of 50 littermates treated with vehicle alone served as controls. A 24-hr pharmacokinetic analysis showed that AG was readily absorbed after i.p. administration, peaked in plasma (9.0 micrograms/ml) at 0.5 hr and had a half-life of 1.88 hr. Steady-state values for the area under the curve for the therapeutic regimen were 20.51 and 16.35 (micrograms)(hr)/ml for the 0000 to 1600 and 1600 to 2400 hr, respectively. In terms of therapy, life-table analysis indicated the frequency of insulin-dependent diabetes mellitus (6/30 AG-treated vs. 11/31 vehicle-treated, P = .25) and insulitis scores (2.0 +/- 1.1 vs. 2.4 +/- 1.2 in nondiabetic AG- and vehicle-treated mice at 32 wk, respectively, P = .20) were similar in both groups. Flow cytometric analysis revealed no quantitative differences in islet infiltrating macrophages, CD4+ or CD8+ T lymphocytes between groups of animals randomly killed at 8, 16 and 32 wk. Although not eliminating a role for nitric oxide in the pathogenesis of insulin-dependent diabetes mellitus, prophylactic treatment with AG did not significantly impact the onset of insulitis or diabetes in nonobese diabetic mice.

Animals↗
PubMed1995

Detection of exocytosis at individual pancreatic beta cells by amperometry at a chemically modified microelectrode.

Amperometry at a carbon fiber microelectrode modified with a composite of ruthenium oxide and cyanoruthenate was used to monitor chemical secretions of single pancreatic beta cells from rats and humans. When the insulin secretagogues glucose, tolbutamide, and K+ were applied to the cell, a series of randomly occurring current spikes was observed. The current spikes were shown to be due to the detection of chemical substances secreted from the cell. Chromatography showed that the primary secreted substance detected by the electrode was insulin. The current spikes were strongly dependent on external Ca2+, had an average area that was independent of the stimulation method, and had an area distribution which corresponded to the distribution of vesicle sizes in beta cells. It was concluded that the spikes were due to the detection of concentration pulses of insulin secreted by exocytosis.

Animals↗
PubMed1995

Azotobacter vinelandii citrate synthase.

We have purified the citrate synthase from Azotobacter vinelandii and have determined that the size of the subunit is 48,000 Da and the structure of the holoenzyme is a hexamer. This contrasts with earlier estimates that indicate a 58,000 Da subunit and a tetrameric structure. In addition, the enzyme is allosteric with a Hill coefficient of 1.5 and is inhibited by NADH. The Hill coefficient is changed to about 1 by high ionic strength and AMP. The enzyme is thus similar to the citrate synthases of many other Gram-negative, facultative, anaerobic organisms. In addition, the amino acid sequence of about 100 residues has been determined and found to be highly similar to the sequence of Pseudomonas aeruginosa citrate synthase.

Amino Acid Sequence↗
PubMed1995

Crystallization and X-ray analysis of a single fab binding domain from protein L of Peptostreptococcus magnus.

Protein L is a multidomain cell wall constituent of certain strains of Peptostreptococcus magnus which binds to the variable domain of immunoglobulin kappa-light chains. A single immunoglobulin-binding domain of M(r) = 9000 from this protein has been isolated and crystallized. The crystals are of space group P4(2)2(1)2, with cell dimensions a = b = 66.9 A, c = 68.3 A, and diffract to at least 2.2 A resolution. The asymmetric unit of the crystal contains two molecules of the protein L domain, related by a noncrystallographic 2-fold axis, as revealed by a self-rotation function calculated with native diffraction data.

Bacterial Proteins↗
PubMed1994

The thymus as a site for evaluating the potency of candidate beta cell autoantigens in NOD mice.

Intrathymic (i.t.) injection of islet cells or whole islets retards development of insulin dependent diabetes mellitus (IDDM) in spontaneous animal models of the disease. Protection of 4-week-old prediabetic NOD/Lt female mice from subsequent IDDM development was specific for the it route of administration since intraperitoneal injection of an equal number of syngeneic islets failed to retard IDDM. The protective effect of i.t. injection of islet cells was compared with the effect of i.t. injection of syngeneic peritoneal exudate cells, NIT-1 cells, bovine serum albumin (BSA), ABBOS peptide, a 52 kDa islet cell membrane protein, various synthetic peptides from human glutamic acid decarboxylase (GAD) and a Coxsackievirus B4-derived peptide with homology to GAD. Interestingly, only a GAD-derived peptide containing sequence homology to Coxsackie-virus B4, and the corresponding Coxsackievirus B4-derived peptide, delayed IDDM onset. To establish the immunological mechanism underlying the reduced IDDM incidence following i.t. injection of islet cells, adoptive transfer of splenic leukocytes into NOD-scid/scid mice was performed. Splenic leukocytes from i.t.-injected non-diabetic females transferred IDDM into NOD-scid/scid recipients, but more slowly than splenocytes from unmanipulated, diabetic (control) donors. Co-transfer of 1:1 mixtures of splenic leukocytes from it islet-injected (and diabetes-free) NOD/Lt females and from untreated NOD/Lt diabetic donors produced IDDM as rapidly as splenocytes from diabetic donors injected alone. Hence, any peripheral suppression generated in i.t.-protected females was not sufficiently strong to prevent IDDM transfer by committed T-effector cells from the diabetic donors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptor Proteins, Signal Transducing↗
PubMed1994

Prevention of diabetes in the NOD mouse: implications for therapeutic intervention in human disease.

The prevention of insulin-dependent diabetes (IDD) in humans remains an elusive goal, despite the broad spectrum of therapeutic interventions that prevent the development of IDD in the non-obese diabetic (NOD) mouse. Can an animal model in which spontaneous autoimmune pathology is interrupted so easily serve as an archetype for the design of clinical trials aimed at the prevention of IDD in humans? In this article, Mark Bowman, Edward Leiter and Mark Atkinson review the intervention strategies that prevent IDD in the NOD mouse and indicate why these studies may well be relevant to the prevention of IDD in humans.

Animals↗
PubMed1994

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin. The four C units were shown to be responsible for binding to immunoglobulin and the four D units for binding to albumin. This protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. The albumin-binding units have high amino acid sequence identity with the albumin-binding units of streptococcal cell-surface proteins. The gene contains three sites available for internal initiation of translation resulting in three active proteins. The protein L molecule presented in this report was compared with a previously reported protein from Peptostreptococcus strain 312. The two proteins differ in several respects, including size and the number and types of repeat units.

Amino Acid Sequence↗