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Query: UMLS:C0033036 (APC)
 10,214 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies on the processing of insulin as an Ag for the presentation to MHC class II-restricted T cells revealed that the amino acid residues 1-14 of the insulin A chain are recognized by insulin-specific T cells. An A1-14 peptide containing three cys-residues that were protected by S-sulfonate groups still needed processing by APC for efficient presentation similar to native insulin. We suspected that reductive deblocking or opening of disulfide bonds that generates CysSH-residues may be an essential processing step for these Ag. Due to the instability of SH-groups it was not possible to test A chain peptides with free SH-groups in the usual way for processing-independent presentation by fixed APC. However, under acidic conditions (pH 5) during APC pulsing with the Ag we could demonstrate that the freshly reduced A1-14 fragment as well as reduced insulin are able to bind to Ia Ag and to stimulate appropriate T cells without further processing. Various substitutions of cys-residues by Ser within this peptide revealed that only CysA7 is critical for Ia binding and/or T cell recognition. In intact insulin, this residue links the A chain containing the T cell epitope to the B chain. Therefore, we propose that insulin processing is not dependent on proteolysis or on the generation of a conformational determinant but on the separation of A and B chains resulting in A chains whose cys-residues are converted into CysSH.
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PMID:In vitro processing of insulin for recognition by murine T cells results in the generation of A chains with free CysSH. 157 64

We have examined mechanisms of tolerance to circulating self-proteins in mice that are transgenic for human insulin. Normal, nontransgenic mice develop serum antibody responses when injected with human insulin in CFA; syngeneic transgenic mice do not. B cell responsiveness was assessed by immunizing with human insulin coupled to a T-independent Ag, Brucella abortus. No differences were found in the numbers of insulin-specific splenic plaque-forming cells between transgenic and nontransgenic mice suggesting that insulin-specific B cells are not tolerant in transgenic mice. Similarly, APC from transgenic and nontransgenic mice display no differences in their ability to process and present human insulin to human insulin-specific T cells in vitro. However, marked differences were detected between transgenic and nontransgenic T cells. Lymph node T cells from transgenic mice primed with human insulin provided no detectable helper activity for secondary antibody responses to human insulin whereas, lymph node T cells from nontransgenic mice did. Nevertheless, lymph node T cells from transgenic mice developed significant proliferative responses to human insulin. Lymph node T cells obtained from transgenic and nontransgenic mice were fused to BW5147 and human insulin-specific T cell hybridomas were generated. The fact that human insulin-specific T cell hybridomas were obtained from the transgenic mice suggests that these T cells were not clonally deleted. In addition, APC from transgenic mice did not stimulate human insulin-specific hybridomas from normal mice in the absence of exogenous insulin. We suggest that T cells specific for human insulin are not deleted in the thymus of transgenic mice because APC in the thymus do not bear the requisite levels of endogenous human insulin/Ia complexes. Therefore, we conclude that tolerance in the transgenic mice is preserved by peripheral mechanisms.
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PMID:A peripheral mechanism preserves self-tolerance to a secreted protein in transgenic mice. 197 65

Brain alpha-adrenoceptor (alpha-AR) binding was examined as a possible explanation for the persistence of diet-induced obesity (DIO) or resistance (DR) in rats after they were returned to chow from a high-energy, fat and sucrose diet (CM diet). Adult Sprague-Dawley rats (n = 28) were fed the CM diet for 12 weeks. Those that gained more weight than chow-fed controls were classified as DIO and those that gained the same weight as controls were called DR. The 10 heaviest DIO and 8 lightest DR rats were then placed on chow for an additional 14 weeks. After the entire 26-week period, the body weights of DIO rats were still 21 per cent greater and those of the DR rats were 9 per cent less than 7 chow-fed controls. DIO retroperitoneal fat pads were also 62 per cent heavier while DR pads were equal to controls. Plasma insulin and glucose levels were comparable in all 3 groups. Receptor autoradiographic studies of brain alpha 1-AR (3H prazosin; PRZ) and alpha 2-AR (3H-paraminoclonidine; PAC) adrenoceptor binding were carried out using these animals at the end of 26 weeks. Binding to alpha 1-AR was comparable in all groups but alpha 2-AR binding was 47-103 per cent higher in DIO compared to DR and chow-fed controls in 14 of 17 brain areas assessed. These included the dorsomedial, ventromedial and paraventricular hypothalamic n. and all amygdalar areas. Such widespread differences in alpha 2-AR binding in rats fed the same diet but of differing body weights suggest that alpha 2-AR binding is a marker for differences in body weight regulation and may be important in the control of the differences in weight gain.
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PMID:Increased brain 3H-paraminoclonidine (alpha 2-adrenoceptor) binding associated with perpetuation of diet-induced obesity in rats. 197 22

Three human T cell lines specific for the A loop of beef insulin were studied to determine the requirements for Ag processing. The data show that the conformation of the A loop of insulin is required for recognition and that the B chain of insulin per se is not necessary for this response. Processing of native insulin was required for responses of all three T cell lines; however, each displayed a different pattern of sensitivity to inhibition of processing and aldehyde fixation of APC. A peptide comprised of two disulfide-linked A chains was partially stimulatory when presented by fixed APC whereas A chain monomers and disulfide-linked A and B chain peptides were not. The response to native insulin, peptides, and A chain dimers was sensitive to chloroquine suggesting that none of these moieties is the terminal processed peptide recognized by insulin immune T cells. The unique patterns of fine specificity, processing requirements, and recognition of aldehyde-fixed antigen-MHC for each T cell line suggest the hypothesis that Ag processing leads to heterogeneity of the T cell repertoire for a single epitope of insulin.
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PMID:Antigen processing and the human T cell receptor repertoire for insulin. 246 May 29

The B chain of mammalian insulins contains appropriately spaced amino acids that predict recognition by T cells. However, all T cell clones from an HLA-DR1, Dw6 diabetic donor recognize epitopes associated with the A chain, and the B chain was found to inhibit these responses. Effective intramolecular competition at the level of the APC, not a direct effect on the T cell, is responsible for the inhibition. Insulin B chain contains two clusters of amino acid homology with the TCR beta chain and B chain peptides lacking these clusters do not compete for antigen presentation. A hole in the repertoire for T cells that recognize this portion of the insulin molecule may arise in the thymus by deletion of T cells that recognize similar peptides.
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PMID:Insulin B chain functions as an effective competitor of antigen presentation via peptide homologies present in the thymus. 247 79

In this study we investigated aspects of antigen processing using insulin and insulin A chain-derived fragments as model antigens in Ab alpha Ak beta-restricted T-cell stimulation. Similarly to other proteins, the immunodominant region of insulin recognized by these T cells is limited in size. It is located on the insulin A chain and encompasses a portion of the molecule that is represented faithfully by peptide A1-14(SSO3-)3. Efficient presentation of intact insulin and its entire A chain is dependent on uptake and processing by APC. Whereas peptides stemming from various globular proteins are known to be presented to T cells by APC without requiring processing, this is not the case with A-chain fragment A1-14 (SSO3-)3. This observation suggested that, in addition to proteolytic degradation, other mechanisms might play a role in the processing of these antigens. Three cys-residues are located in close proximity to those amino acid residues of the insulin A chain that are inferred to participate in the specific interaction with MHC class II molecules and the TcR. In A-chain derivatives that are stimulatory for the T cells or in intact insulin these cys residues are engaged in disulfide bonds or are S-sulfonated. Both linkages can be reversibly modified by reaction with thiols. Functional data indicate that from intact insulin and from structurally distinct A-chain derivatives a closely similar or identical peptide is formed and bound to class II molecules for recognition by the T cells. The question arises as to whether, in this processed peptide, the cys residues are present in reduced form, engaged in disulfide bonds, or are modified in some other way. Taken together, these findings suggest that modification of cys residues or isomerization of disulfide bonds may play a role in insulin processing. It can be expected that other proteins carrying cys residues in their immunodominant peptides may show similar processing requirements. The inhibition of N-glycosylation of proteins by tunicamycin in APC blocked the processing and presentation of insulin and OvA whereas, under the same conditions, the presentation of a processing-independent peptide was not affected. Furthermore, an autoreactive T-cell clone was capable of recognizing tunicamycin-treated APC. Since the expression of class II molecules was found to be unaltered as demonstrated by cytofluorometric analysis the deficient N-glycosylation appears to have little influence on class II antigen-mediated T-cell recognition but interferes with uptake of antigen and/or its processing by APC.
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PMID:Processing requirements for the recognition of insulin fragments by murine T cells. 247 28

To study the biochemistry of processing of a soluble protein Ag by an APC, we investigated how 125I-labeled human insulin (HI) is processed in situ by TA3 mouse hybridoma B cells. Fractionation of TA3 cells into their extracellular, plasma membrane-associated and intracellular compartments coupled with the use of HPLC enabled us to analyze several peptides derived from each compartment. One HI peptide found in all three compartments is composed of residues A1-A14 disulfide-linked to B7-B26 (A1-A14/B7-B26). The presence of this peptide in the extracellular compartment likely resulted from digestion of HI by an enzyme(s) released from the APC. Extracellular processing of radiolabeled HI was inhibited completely by unlabeled HI and N-ethylmaleimide, an inhibitor of a previously described insulin-specific protease, partially by lysozyme but not by BSA or OVA. This suggests that the enzyme involved in the extracellular processing of insulin is relatively insulin-specific and gives rise to the A1-A14/B7-B26 peptide. The processing of HI both at the plasma membrane and intracellularly was inhibited by chloroquine, monensin, and NH4Cl, suggesting that both intracellular pH changes and endocytic and exocytic events may be required for these compartments to process insulin. Kinetic analyses revealed that the processing of insulin into the A1-A14/B7-B26 peptide is first detected at the plasma membrane then intracellularly and finally in the extracellular compartment. This unlabeled A1-A14/B7-B26 peptide was purified from the extracellular compartment of TA3 APC by HPLC; when presented by TA3 APC this peptide effectively stimulated pork insulin (PI/I-Ad) specific Th cells to secrete IL-2. These data, taken together with the identification of another processed insulin peptide, A7-A11/B7-B26, have enabled us to elucidate the first steps in the biochemical pathway(s) of processing of insulin as an Ag in a B cell APC.
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PMID:Processing and presentation of insulin. II. Evidence for intracellular, plasma membrane-associated and extracellular degradation of human insulin by antigen-presenting B cells. 265 61

Current models suggest that Ag undergoes proteolytic cleavage in APC and that resultant peptide fragments associate with class II histocompatibility glycoproteins before recognition by helper T cells. Little direct information is available concerning the physical structure and membrane association of Ag processed under physiologic conditions. A model system, employing a series of biotinylated insulin derivatives, was used to examine the domains of Ag that are presented by APC. We reasoned that avidin should block the response of T cells to a given derivative only if biotin is retained on the functionally relevant form of Ag after processing. By utilizing derivatives modified at selected sites one should be able to determine whether specific sites remain after processing. By using F1 APC pulsed with biotinyl-insulin derivatives modified through the free amino groups of the A1, B1, or B29 amino acids, and T cell hybridomas restricted to I-Ad or I-Ab, we found that avidin inhibited the I-Ad-restricted response to A1, but not B1 or B29 derivatives. By contrast, specific inhibition of the I-Ab-restricted response was observed by using all three derivatives. These results suggest that the processed form of insulin recognized in association with I-Ab is largely intact and includes residues from both chains (A1, B1, and B29). The differential inhibition observed by using T cells restricted to different class II alleles demonstrates that processed Ag associated with I-Ab differs in conformation or structure from that associated with I-Ad. This experimental approach should prove valuable in characterizing the actual structure of processed Ag recognized by T cells.
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PMID:Probing the structure of processed antigen by using biotin and avidin. MHC-dependent inhibition of responses to selected biotinyl-insulin derivatives. 268 18

Recent studies indicate that Ag pass through a chloroquine-sensitive intracellular pathway in accessory cells before they are recognized by class II-restricted T cells. Our results indicate that this is also true for insulin. Unexpectedly, we find that protein synthesis is required for optimal accessory cell-dependent processing of insulin and other proteins by adherent macrophages. Treatment of APC with inhibitors of protein synthesis, before and during exposure to Ag, inhibits their subsequent ability to activate murine T cell hybridomas. Experiments are described which suggest that this effect is localized to intracellular processing of Ag rather than uptake or presentation, per se. Inhibition is reversible, and is not observed in special situations where intracellular processing of Ag is not required. A distinct lag period is required for inhibition of processing after inhibition of macrophage protein synthesis. One possible interpretation is that protein synthesis is necessary for maintenance of a labile protein crucial for intracellular processing of Ag. Alternatively, the susceptibility of processing to inhibitors of protein synthesis may reflect an obligate intracellular association of Ag and newly synthesized class II histocompatibility molecules.
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PMID:Protein synthesis in antigen processing. 304 13

An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence. The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion. Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques. The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity. A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart. Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min. These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner.
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PMID:Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli. Kinetics of processing by antigen presenting cells. 307 Mar 57


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