Induction of autoimmunity by immunization with hapten-modified hen egg lysozyme in hen egg lysozyme-transgenic mice

To understand the mechanism of autoimmunity induction, hen egg lysozyme (HEL)-transgenic (Tg) C57BL/6 (B6) mice were immunized with HEL or phosphorylcholine-conjugated HEL (PC-HEL). Repeated immunization of HEL-Tg mice with native HEL failed to induce the antibody response against HEL. However, immunization with PC-HEL generated a significant anti-HEL antibody response. Immunization of the Tg mice with dominant (HEL(74-88)) or cryptic (HEL(47-61)) T-cell epitope peptide stimulated the corresponding T-cell response and similarly yielded the anti-HEL antibody response. Predominance of immunoglobulin G1 (IgG1) anti-HEL antibody response in the HEL-Tg mice and preferential IL-4 production by HEL-specific T cells suggested the dependency of the antibody response to the presence of T helper 2. HEL-Tg mice received HEL-primed B6 T cells, but not HEL-primed Tg T cells, were able to generate anti-HEL antibody response following PC-HEL immunization. The pattern and the level of epitope peptides generated by splenic antigen-presenting cells indicated that PC-HEL results in much more efficient processing as compared to HEL. These results strongly suggest that the enhancement of antigen processing by hapten (PC) conjugation to the antigen facilitates more efficient stimulation of T cells reactive to self antigen, HEL in HEL-Tg mice resulting in the production of anti-self HEL antibody.     

Quantification of hen egg white lysozyme in cartilage by an enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) with an inhibition step to quantify hen egg white lysozyme (HEWL) on a weight basis. The assay is relatively insensitive to changes in ionic strength or pH. Quantification is not affected by the presence or large amounts of mammalian lysozymes or inhibitors of enzymatic activity such as soluble chitin oligomers. We used this ELISA to show that the HEWL content of chick cartilages increases progressively between days 14 and 18 of embryonic life. The maturation-related increase does not appear to be the result of increased synthesis by the chondrocytes for the latter did not synthesize detectable amounts of lysozyme in vitro. Evidence was obtained to suggest that most, if not all, the lysozyme in cartilage is derived from the surrounding fluids which come in contact with the matrix.     

Analysis of hen egg white lysozyme (HEL)-specific delayed type hypersensitivity hybridomas

Hen egg white lysozyme (HEL) specific delayed type hypersensitivity hybridomas (TDTH hybridomas) were established by fusing HEL specific C3H/HeN (H-2k haplotype) TDTH with BW5147 thymoma cells. TDTH hybridomas, which had Lyt-1+2- and Thy-1.2 antigens on their surfaces, were injected into mouse footpads with HEL antigens, and foot pad swellings were measured 24 h later. Mice with I-Ak haplotype showed strong responses, and their swellings were reduced by anti-I-Ak specific monoclonal antibodies, which suggests that the activity of the hybridomas is restricted by I-Ak. However, TDTH hybridomas did not modulate HEL specific T-cell proliferation responses.     

Cell type-specific processing of the I-Ed-restricted hen egg lysozyme determinant 107–116

Class II major histocompatibility complex heterodimers present to T cells determinants as sets of antigen fragments with ragged N and C termini. It is not yet elucidated whether different types of antigen-presenting cells generate identical sets of peptides containing the same determinant. Taking advantage of recombinant I-E^d molecules produced by insect cells as empty heterodimers, a sensitive T cell stimulation assay was developed to analyze naturally processed hen egg lysozyme (HEL) peptides. I-E^d preparations were isolated from antigen-presenting cells cultured with HEL. Following acid treatment, peptides eluted from I-E^d were chromatographed and the fractions incubated at acidic pH with purified recombinant I-E^d molecules, conditions which favor peptide binding. The stimulatory capacity of the reconstituted peptide-I-E^d complexes adsorbed on the well surface of cell culture plates was then evaluated by measuring interleukin-2 secreted by an HEL 107–116-specific, I-E^d-restricted T cell hybridoma. We found that the B lymphoma A20 and an I-E^d-transfected fibroblast cell line generated distinct sets of peptides containing the HEL sequence 107–116. Our results suggest the possibility that presentation of one determinant by different types of antigen-presenting cells stimulates populations of T cells with distinct fine antigen specificities.     

Sequential appearance of antibodies directed against different antigenic determinants of hen egg-white lysozyme

Three rabbits immunized with hen egg-white lysozyme (three injections in complete Freund's adjuvant) were bled weekly for a period of 4 months. Each individual antiserum was tested for the presence of antibodies specific towards both native lysozyme and two distinct antigenic determinants of the molecule, comprising the “loop” peptide (residues 60-83 containing one disulfide bond) and a fragment containing the amino and carboxy termini of lysozyme. Variations were observed in the amounts and the time of appearance of antibodies with different specificities. The first demonstrable antibodies, 10 days after the onset of immunization, were antibodies directed towards intact lysozyme. At this time, no antibodies to the two isolated antigenic fragments of the molecule could be detected. Antibodies specific for these two determinants appeared at much later bleedings 2-3 months after immunization. Although the general pattern of the induction of antibodies with different specificities was the same in all three rabbits, individual differences were never-theless observed among the three animals used in this study.     

Endotoxin-neutralizing activity of hen egg phosvitin

Endotoxin, also known as lipopolysaccharide (LPS), is responsible for initiating host responses leading to inflammation and sometimes unwanted sepsis, which is associated with high mortality in patients. No therapeutic agents to date are efficacious enough to protect patients from LPS-mediated tissue damage and organ failure. Previously, egg yolk protein phosvitin (Pv) in zebrafish has been shown to act as a pattern recognition receptor, capable of binding to the microbial cell wall components including LPS, we therefore wonder if it has the capacity to block LPS toxicity. In this study we first demonstrated that hen Pv, a naturally occurring protein rich in egg yolk, had antimicrobial activity against Escherichia coli and Staphylococcus aureus under thermal stress, and then showed that Pv was able to bind to LPS, lipoteichoic acid and peptidoglycan as well as the microbes E. coli and S. aureus. More importantly, we revealed that Pv significantly inhibited LPS-induced tumor-necrosis factor (TNF)-α release from murine RAW264.7 cells and considerably reduced serum TNF-α level in mice. Additionally, hen Pv could promote the survival rate of the endotoxemia mice. Furthermore, hen Pv displayed no cytotoxicity to murine RAW264.7 macrophages and no hemolytic activity towards human red blood cells. Taken together, these data suggest that Pv is an endotoxin-neutralizing agent with a therapeutic potential in clinical treatment of LPS-induced sepsis.     

Use of defined estrone glucuronide-hen egg white lysozyme conjugates as signal generators in homogeneous enzyme immunoassays for urinary estrone glucuronide

Three structurally characterized estrone glucuronide-lysozyme conjugates, E1 (a 60:40 mixture acylated at K3 and K97), E3 (acylated at K33), and E5 (acylated at both K33 and K97) were isolated and purified using a combination of cation-exchange chromatography on S-sepaharose in 7M urea and hydrophobic interaction chromatography on butyl sepharose. Urea was essential to separate the conjugates into six chromatographically homogeneous fractions. In the absence of urea, complex mixtures of lysozyme and the six conjugate fractions were always encountered. The E1, E3, and E5 conjugates were highly inhibited by a sheep polyclonal anti-estrone glucuronide antibody only after the hydrophobic interaction chromatography step. The high level of inhibition enabled all three conjugates to be utilized as signal generators in homogenous enzyme immunoassays for urinary estrone glucuronide. Despite the apparently higher affinity of E3 for the antibody, both E1 and E3 gave standard curves that were indistinguishable provided that 1.7-fold more antiserum was used for E1. Both E1 and E3 yielded menstrual cycle urinary data that agreed with that provided by the Ovarian Monitor pre-coated assay tubes. Although, the menstrual cycle pattern was similar for the three signal generators, the E1G excretion rates yielded by E5 as the signal generator were only 60% of the reference values. Despite structural differences, there was no advantage gained in separating E1 and E3, but higher substituted conjugates such as E5 need removal for best assay performance.     

Processing and presentation of hen egg-white lysozyme by macrophages

The processing and presentation by macrophages of the well-defined protein hen egg-white lysozyme (HEL) was analyzed using two HEL-specific T cell hybridomas. The processing studies revealed that both clones required that native HEL be processed, while neither clone required any processing of a tryptic digest of lysozyme. A differential requirement for processing was found for the intact, denatured lysozyme (CM-HEL) with one clone (2A11) requiring processing, and a second clone (3A9) did not require any processing. The determinant on the HEL molecule that both clones recognized was localized to a tryptic fragment containing residues 46 to 61. By testing the immunogenicity of fragments of the 46-61 peptide, mouse lysozyme, and human lysozyme, we were able to localize the T cell determinant to either of two residues, Gly-49 or Leu-56.     

Comparison of day 3 FSH serum values as determined by six different immunoassays

BACKGROUND: The accurate assessment of FSH concentration is important for evaluating ovarian function prior to IVF. However, a number of different assay techniques are currently in use, leading to inconsistencies in the hormone data being reported. To address this problem, we measured FSH concentration using a number of commercially available systems. METHODS: Day 3 serum FSH levels were measured in 215 healthy fertile women using six different immunoassays: Coatria (125)I (Bio-Mérieux), ACS-180 (Bayer Diagnostics), Advia-Centaur (Bayer Diagnostics), Vitros ECi (Ortho-Clinical Diagnostics), Architect i2000 (Abbott) and Elecsys 2010 (Roche Diagnostics). RESULTS: According to the immunoassay, means +/- SD of FSH concentrations were: 6.5 +/- 2.2 mIU/ml for Coatria (125)I, 6.8 +/- 2.7 mIU/ml for Advia-Centaur, 6.7 +/- 3.0 mIU/ml for Vitros ECi, 7.6 +/- 3.0 mIU/ml for ACS-180, 8.2 +/- 3.3 mIU/ml for Architect i2000 and 8.8 +/- 3.0 mIU/ml for Elecsys 2010. CONCLUSION: Day 3 FSH values determined by six different immunoassays were significantly different (P < 0.01, paired t-test). Physicians must take care when interpreting results from different clinical laboratories.     

Immunodominant protein epitopes II. The primary antibody response to hen egg white lysozyme requires and focuses upon a unique N-terminal epitope

The antibody response to a defined protein antigen, hen egg white lysozyme (HEL) has been investigated using an aminopeptidase-treated HEL molecule, des-1,2,3-HEL (AP-HEL). Surprisingly, removal of these three N-terminal residues eliminates an epitope which is a dominant B cell determinant recognized in the primary antibody response to HEL. Thus, the initial antibody response focuses on a very small region of the molecule. Even more striking is the observation that removal of this epitope markedly reduces the immunogenicity of HEL. Therefore, the epitope is not only the focus of the primary antibody response, but is essential for the initiation of the response. This report demonstrates that a selective mechanism must be activated during the response to this protein antigen. Of the multitude of B cell determinants present on HEL, only a limited number are focused upon by the immune system.     

Structural relationships between carrier epitopes and antigenic epitopes on hen egg-white lysozyme.

The spatial relationship for T-B cell cooperation between antigenic epitopes and carrier epitopes on the antigen molecule was studied. Two mono-DNP substituted derivatives of hen egg-white lysozyme (HEL), DNP1-33HEL and DNP1-96HEL were used as antigens; the former is dinitrophenylated only at lysine-33 and the latter only at lysine-96. Fragment peptides of HEL were used to induce specific T cells to the respective sites of the antigen. Adoptive cell-transfer experiments of site-specific T cells and DNP-primed B cells directly showed that multiple distinct carrier epitopes for T cells could help the antibody responses to the single antigenic epitope for B cells and that a single carrier epitope could help antibody responses to multiple antigenic epitopes. T cells primed with a synthetic peptide SP34-54 (corresponding to sequence 34-54 of HEL) co-operated with DNP-primed B cells on challenge with DNP1-96HEL, but not with DNP1-33HEL.     

Rapid method of extraction of antibodies from hen egg yolk

Antibodies were raised in laying hens and isolated from the yolk of their eggs by precipitation with precooled (-20 degrees C) propane-2-ol and removing lipid material with propane-2-ol and acetone. The dried precipitate was extracted with phosphate buffer and shown to contain IgG antibodies and a small amount of additional protein. By Ouchterlony gel diffusion and rocket immunoelectrophoresis, the concentration and quality of specific antibodies in the extracts were comparable to those found in the serum of rabbits or to the IgG separated from yolk by other methods. The new isolation procedure is rapid, reliable and convenient.     

Cytosolic targeting of hen egg lysozyme gives rise to a short-lived protein presented by class I but not class II major histocompatibility complex molecules

A way to study the role of intracellular trafficking of an antigen in its presentation to T cells is to target the antigen to various cell compartments of the antigen-presenting cells (APC) and compare the nature of the complexes associating major histocompatibility complex (MHC) molecules and antigenic peptides, expressed on the cell surface. MHC class I+ and MHC class II+ mouse L fibroblasts secreting hen egg lysozyme (HELs cells) or expressing HEL in their cytosol (HELc cells) were obtained after transfection with HEL cDNA and signal sequence-deleted HEL cDNA, respectively. HEL was evidenced in both HELs- and HELc-transfected cells and the former type of transfectant secreted a large amount of HEL. However, HEL produced in the cytosol exhibited a short half-life of less than 5 min. HEL-derived peptides could not be shown biochemically either in HELc- nor in HELs-transfected cells. We then studied the capacity of these cells to present HEL to HEL-specific class I- and class II-restricted T cells. Both cell types could be recognized by the HEL-specific MHC class I-restricted CTL clones. In contrast, MHC class II-HEL peptide complexes, recognized by HEL-specific helper T cell hybridomas, could be detected on MHC class II+ HELs- but not HELc-transfected cells. In vivo experiments showed, however, that HELc-transfected cells could provide host APC with HELc-derived peptides able to associate with MHC class II molecules. This was inferred from the capacity of MHC class II-HELc-transfected cells, unable by themselves to elicit any anti-HEL antibody response, to prime syngeneic and allogeneic mice against HEL. The priming was revealed by the induction of an antibody response after a boost with an amount of HEL unable itself to elicit an antibody response.     

Constraints in antigen processing result in unresponsiveness to a T cell epitope of hen egg lysozyme in C57BL/6 mice.

T cell hybridoma clones were derived after fusion of BW-5147 parent cells with lymphocytes from C57BL/6 mice injected with phosphorylcholine (PC)-hen egg lysozyme (HEL) conjugates. Several T cell hybridomas were preferentially reactive with PC-HEL over unconjugated HEL, and a particular clone (PC-H4.1) was further analyzed. This T cell hybridoma clone could respond to its maximal level toward unconjugated HEL only when the dose of HEL was increased to 5-10-fold of the PC-HEL concentration. Interestingly, this clone was not stimulated by unfolded HEL (by S-carboxymethylation) to the level of PC-HEL. A synthetic peptide representing the amino acid position 47-61 of HEL, which is known to be non-immunogenic upon HEL injection in C57BL/6 mice, was able to stimulate the hybridoma only to a level comparable to that induced by unconjugated HEL. The T cell response to this synthetic peptide required an additional antigen-processing step, based on its inability to stimulate T cells after treatment of antigen-presenting cells with leupeptin, chloroquine or paraformaldehyde. Deletion of a single C-terminal amino acid residue of HEL 47-61 (arginine) significantly enhanced (10-100-fold of HEL 47-61) the T cell reactivity and abrogated the necessity of further antigen processing. These results suggest that the lack of a T cell response to a certain epitope may not be due to the lack of a T cell repertoire reactive to the epitope. In some cases, the unresponsiveness may be due to the difficulty in generating the particular epitopes. Taken together, modification of the lysozyme molecule with PC conjugation may facilitate further antigen processing of HEL to generate an optimal epitope for the nonresponder mice.     

Processing and presentation of intact hen egg-white lysozyme by dendritic cells.

Dendritic cells in lymphoid tissues are of key importance as highly specialized antigen-presenting cells for the induction of T lymphocyte responses. Conflicting results have been published regarding antigen processing of intact proteins by dendritic cells. We now report that highly purified dendritic cells isolated from H-2k mouse spleens very efficiently generated immunogenic fragments of intact hen egg-white lysozyme (HEL) protein to present to an I-Ak-restricted T hybridoma cell line, specific for HEL peptide 46-61. Dendritic cells required 100 times less HEL protein than lipopolysaccharide-induced B cell blasts for effective presentation. Uptake of 125I-labeled HEL protein by dendritic cells and inhibition of presentation of HEL protein by chloroquine treatment was observed. This indicates an endocytotic process and the involvement of acidified compartments. Since the supernatant of dendritic cells, that were incubated with intact HEL protein, contained immunogenic fragments, further evidence for processing of HEL protein by dendritic cells was obtained. When HEL protein was covalently coupled to beads, dendritic cells were not able to ingest these beads, but could still process HEL protein for presentation. This suggests cell surface processing of HEL protein, although internalization of HEL protein released from the beads cannot be excluded. Taken together, these data show that H-2k dendritic cells are capable of processing and presenting intact HEL protein.     

Immunodominant protein epitopes. I. Induction of suppression to hen egg white lysozyme is obliterated by removal of the first three N-terminal amino acids

The lack of response to hen egg white lysozyme (HEL) by C57BL (H-2b) mice has been demonstrated previously to be related to the induction of suppressor T (Ts) cells which recognize the amino terminal region of HEL. In this report, the nature of the protein determinant required for Ts cell induction is more precisely detailed using des-1,2,3-HEL (AP-HEL) prepared with an aminopeptidase purified from Aeromonas proteolytica . Remarkably, the removal of just these three amino acids obliterates the ability of HEL to induce Ts cells specific for HEL. Additionally, in contrast to HEL, AP-HEL is able to prime for an in vitro T cell proliferative response to either AP-HEL or HEL. Thus, removal of a very limited region of a protein antigen can drastically alter its immunogenic properties.     

Comparison of different sandwich enzyme immunoassays for the quantitation of human apolipoproteins A-I and A-II

Variants of a non-competitive enzyme immunoassay for human apolipoproteins A-I and A-II are described. The method developed is highly sensitive and highly specific. There is a good correlation between the reference technique (electroimmunoassay) and both apolipoprotein A-I and A-II assays (r=0.92). For the apolipoprotein A-I assay, seven simple treatments were studied. Two of these treatments (plasma plus Tween 20 and serum plus apo A-II) greatly increased the precision and the accuracy of the results. For the apo A-II measurement, no treatment of the samples is necessary, but plasma should be preferentially used rather than serum.     

Comparison of five different immunoassays for the detection of Borrelia burgdorferi IgM and IgG antibodies

The performances of five commercially available enzyme immunoassays were compared for the detection of Borrelia burgdorferi IgM and IgG antibodies. Sensitivity was assessed with European serum samples collected from 45 patients with clinically defined Lyme disease in conjunction with a positive immunoblot (n = 44) or other serological test (n = 1). Sensitivities for the detection of IgM and IgG with each test were: Dako IgM 64%; Dako IgG 53%; Serion IgM 89%; and Serion IgG 88%. The Immunetics assay makes no distinction between IgM and IgG antibodies and had a sensitivity of 91%. Specificity was calculated by testing a control group comprising 40 patients with acute Epstein-Barr virus infection, cytomegalovirus infection, syphilis or rheumatoid factor positivity. The specificities achieved for each test were: Dako IgM 78%; Dako IgG 100%; Serion IgM 52%; Serion IgG 92%; and Immunetics 92%. The discriminatory power between control and patient samples appeared highest for the Immunetics assay. Between-run variation was comparable for the five tests and did not exceed 13%. When the Immunetics assay was used as an initial screening test, with low-titre positive results confirmed by an immunoblot, a sensitivity of 91% and a specificity of 100% were achieved. To attain maximal sensitivity, the Serion IgM and IgG tests were also performed on samples with negative Immunetics results. All positive Serion IgM and IgG results were also confirmed by immunoblot. In conclusion, the Immunetics assay, based on a synthetic C6 peptide, can be used reliably as an initial screening test for the serodiagnosis of Lyme disease.     

溶菌酶对头孢他啶诱导的细菌内毒素释放的抑制作用

目的研究溶菌酶对头孢他啶诱导的细菌内毒素（ＬＰＳ）释放抑制作用。方法头孢他啶作用于对数生长期的Ｅ．ｃｏｌｉＯ１１１∶Ｂ４,ＳａｌｍｏｎｅｌａｔｙｐｈｉｍｕｒｉｕｍＬＴ２或Ｅ．ｃｏｌｉＪ５,培养液中含或不含溶菌酶。抗菌素作用３小时后,收集培养上清,测定释放到上清液中的内毒素浓度;将培养上清加入到巨噬细胞中,测定ＴＮＦ－α和ＩＬ－６诱生能力。结果头孢他啶５０μｇ／ｍｌ可引起３种细菌迅速溶解,并导致较高浓度的内毒素释放到培养上清液中,该上清液在体外培养的巨噬细胞上和小鼠体内诱导大量的肿瘤坏死因子（ＴＮＦ－α）和白细胞介素６（ＩＬ－６）产生;溶菌酶可阻止头孢他啶引起的细菌溶解,但不影响其杀菌效力,同时明显降低培养上清中的内毒素浓度;并降低对巨噬细胞炎性因子的诱导能力。结论溶菌酶能抑制头孢他啶诱导的细菌内毒素释放     

Interleukin 12 alters the isotype-restricted antibody response of mice to hen eggwhite lysozyme

Protein antigens elicit humoral responses in mice that consistpredominantly of IgG1 antibodies. We have now investigated theability of IL-12, a cytoklne reported to augment IgG2a antl-haptenresponses through activation of T_h1 cells, to alter antibodyresponses to hen eggwhite lysozyme (HEL). The normal responseof BALB/c mice to HEL is highly restricted to lgG1 expressionand therefore provides an excellent system for determining effectsof cytoklnes on expression of other isotypes. Seven days afterimmunization, IL-12-treated mice demonstrated greatly elevatedHEL-speciflc IgG2a antibody levels and suppressed IgG1 production,while PBS-treated control mice showed a typical lgG1-restrictedresponse. On day 28, IL-12-treated mice showed heightened serumantibody levels of both isotypes. Delaying cytoklne treatmentuntil after the typical IgG1 anti-HEL response had already beenestablished also led to significant elevation of serum IgG2aantibody levels. These effects correlated with increased IFN-production; however, administration of IL-12 plus anti-IFN-had little influence on lgG2a enhancement, although it did relievethe early lgG1 suppression. Furthermore, the differential effectsof IL-12 on Isotype expression did not correlate with time;in fact, IgG2a enhancement correlated with loss of IgG1 suppression.Our findings indicate that (I) IL-12 reproduclbly Induces largeamounts of IgG2a HEL-speclflc antibodies In vivo; (II) it canalter isotype profiles of both primary and secondary responses;and (III) its effects on humoral immunity are not completelyexplained by Induction of T_h1 cell-derived IFN-.