Paramphistomum cervi: antigenic profile of adults as recognized by infected cattle sera

An antigenic profile of adult Paramphistomum cervi was revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from cattle naturally infected with P. cervi, Fasciola gigantica and strongylids. SDS-PAGE of whole worm extracts exhibited 26 distinct protein bands. Immunoblotting analysis of these proteins showed five major antigenic bands which were recognized by serum of individual cattle naturally infected with P. cervi. These antigenic proteins had molecular weights ranging from 23 to 116kDa. One antigenic protein with a molecular weight of 52kDa exhibited a consistent reaction with sera from all infected cattle. It's diagnostic sensitivity, specificity and accuracy using this test were 100%, 98% and 98.9%, respectively. The positive and negative predictive values were 97.6% and 100%, respectively. This finding suggests that the 52kDa protein may be a diagnostic antigen for paramphistomosis.     

Identification of antigenic epitopes recognized by Mac-2 binding protein-specific cytotoxic T lymphocytes for use in cancer immunotherapy

We have previously reported that 90K/Mac-2 binding protein (M2BP) was highly expressed in lung cancer and that M2BP-specific immunity was observed in many of cancer patients. In this study, we analyzed the ability of 11 M2BP-derived oligopeptides with an HLA-A*0201-binding motif to induce M2BP-specific cytotoxic T lymphocytes (CTL) from peripheral blood lymphocytes of normal donors by in vitro stimulation. One of the CTLs that were induced using M2BP216-224 (RIDITLSSV) produced interferon-gamma in response to HLA-A2-positive T2 cells pulsed with the same peptide and lysed MDA-MB-231 cells expressing both M2BP and HLA-A2. The cytolytic activities were blocked by antibodies against HLA class I or CD8. These findings suggest that M2BP216-224 is naturally processed from the native M2BP in cancer cells and recognized by M2BP-specific CTLs in an HLA-A2 restriction. We first identified M2BP-derived CTL epitopes that may be useful as a target antigenic epitope in clinical immunotherapy of cancer.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

Identification of new centrosome proteins by autoimmune patient sera

Compared to other subcellular organelles, centrosome proteome can hardly be studied, due to the difficulties in separation and purification of centrosome. Auto-antisera from 6 autoimmune patients, which recognized centrosome specifically in immunofluorescence, were used to identify the corresponding centrosomal proteins. The sera were first tested by Western blot on whole cell lysate, and all bound antibodies were then eluted from each single band in Western blot membrane to assure which antibody was responsible for the centrosome specific immunofluorescence staining. The corresponding proteins were obtained by immunoprecipitation and identified by mass spectrometry. Six centrosomal proteins, including 2 known centrosomal proteins and 4 proteins with unknown localization or reportedly non-centrosomal localization, were identified. These proteins apparently involve in cell cycle regulation, signal transduction pathways, molecular chaperons, and metabolism enzymes, which may reflect the expected functional diversity of centrosome.     

Proteomic analysis of cancer stem cells in human prostate cancer cells

Results from recent studies support the hypothesis that cancer stem cells (CSCs) are responsible for tumor initiation and formation. Here, we applied a proteome profiling approach to investigate the mechanisms of CSCs and to identify potential biomarkers in the prostate cancer cell line DU145. Using MACS, the DU145 prostate cancer cell line was isolated into CD44+ or CD44− cells. In sphere culture, CD44+ cells possessed stem cell characteristics and highly expressed genes known to be important in stem cell maintenance. In addition, they showed strong tumorigenic potential in the clonogenic assay and soft agar colony formation assay. We then analyzed and identified proteins that were differentially expressed between CD44+ and CD44− using two-dimensional gel electrophoresis and LC-MS/MS. Cofilin and Annexin A5, which are associated with proliferation or metastasis in cancer, were found to be positively correlated with CD44 expression. These results provide information that will be important to the development of new cancer diagnostic tools and understanding the mechanisms of CSCs although a more detailed study is necessary to investigate the roles of Cofilin and Annexin A5 in CSCs.     

Processing of proteins in autophagy vesicles of antigen-presenting cells generates citrullinated peptides recognized by the immune system

Our laboratory has been investigating for some time the nature of the response of T lymphocytes in autoimmunity in the reactions against self-proteins that result in a number of diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis (RA) and others. T cells recognize peptides generated from proteins that are processed by antigen-presenting cells (APC). The peptides may derive from exogenous proteins or from the normal catabolism of self-proteins. The peptides complexed to major histocompatibility complex (MHC) molecules constitute the chemical entity that is engaged by the antigen-receptor of T cells. An important hypothesis postulates that self-peptides that suffer post-translational modifications in the APC may form neo-antigens that are recognized by the immune system and form the target of autoimmunity. Our interest in citrullination in the context of antigen processing and presentation stemmed from studies suggesting that an immune response to citrullinated self-peptides may be involved in autoimmunity. In a first publication, we found T cells that specifically recognized citrullinated peptides after immunization of inbred mice with standard foreign proteins. We used the small protein hen-egg white lysozyme. These T cells only recognized the citrullinated peptide and not the unmodified one, thus proving that a neo-epitope had been created by this modification. But how this modification took place was not known. Our recent report describes a central role for autophagy in citrullination of peptides by APC.     

Processing of proteins in autophagy vesicles of antigen-presenting cells generates citrullinated peptides recognized by the immune system

Our laboratory has been investigating for some time the nature of the response of T lymphocytes in autoimmunity in the reactions against self-proteins that result in a number of diseases, such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis (RA) and others. T cells recognize peptides generated from proteins that are processed by antigen-presenting cells (APC). The peptides may derive from exogenous proteins or from the normal catabolism of self-proteins. The peptides complexed to major histocompatibility complex (MHC) molecules constitute the chemical entity that is engaged by the antigen-receptor of T cells. An important hypothesis postulates that self-peptides that suffer post-translational modifications in the APC may form neo-antigens that are recognized by the immune system and form the target of autoimmunity. Our interest in citrullination in the context of antigen processing and presentation stemmed from studies suggesting that an immune response to citrullinated self-peptides may be involved in autoimmunity. In a first publication, we found T cells that specifically recognized citrullinated peptides after immunization of inbred mice with standard foreign proteins. We used the small protein hen-egg white lysozyme. These T cells only recognized the citrullinated peptide and not the unmodified one, thus proving that a neo-epitope had been created by this modification. But how this modification took place was not known. Our recent report describes a central role for autophagy in citrullination of peptides by APC.     

Immunoblotting identifies and antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera

In SDS-PAGE the immune complexes (IC) of kala-azar patient sera showed intense bands at 55 kDa and 20 kDa corresponding to heavy and light chains of immunoglobulins. In immunoblot experiment, kala-azar and normal IC after treatment with patient sera showed multiple bands of which the band at 55 kDa was most prominent in kala-azar IC. It is known that in kala-azar sera antihuman IgG is present, so the heavy band at 55 kDa region may be due to higher amount of IgG and/or other antigen(s) present at that region. Immunoblot experiments of kala-azar IC with anti gp63 also developed a major band at 55 kDa. It suggests that the antigen (55 kDa) and gp63 have common antigenic epitope (s). Normal IC did not react with anti gp63 indicating absence of this antigen in normal IC. Antigenic similarity between the IC antigen (55 kDa) and gp63 indicated that the former antigen may have been processed from gp63. In summary, identification of a parasite antigen (55 kDa) in IC of kala-azar patients sera may be useful in developing a serodiagnostic assay for visceral leishmaniasis. (Mol Cell Biochem130: 11–17, 1994)Abbreviations IC Immune Complexes - PEG Polyethylene Glycol (Mol wt 8000) - PBS Phosphate Buffer Saline - VL Visceral Leishmaniasis - AVL American Visceral Leishmaniasis - IgG Immunoglobulin G - TBS Tris Buffer Saline - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - gp63 A leishmanial surface glycoprotein of molecular mass 63,000 - TEMED N,N,N,N-Tetramethylethylenediamine     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Two-dimensional gel analysis of proteins in cell lines from the central nervous system of larvalDrosophila

Summary High resolution two-dimensional gel electrophoresis was used to quantitatively analyze the patterns of protein synthesis in three different clones of a nerve cell line (ML-DmBG2) ofDrosophila melanogaster. When patterns of pulse-labeled proteins of the three different clones were compared, I observed quantitative variations affecting the rate of synthesis by twofold or more in 25–30% of the polypeptides and qualitative differences, always affecting less than 2% of the polypeptides. Patterns of protein synthesis were analyzed during the 24 d of culture, revealing both quantitative (increase or decrease; 40%) and qualitative (presence or absence; 3%) differences. More than 70 proteins synthesized in these cultures were secreted into the medium. Among them were two major groups of acidic proteins which disappeared with culture time. When cell lines and intact central nervous systems were compared, large differences in protein synthesis were observed. In fact, only 20% of the synthesized proteins were common to both isolated cells grownin vitro and the original nervous systemin vivo.     

Occurence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen

Abstract An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia -specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occuring in patient sera. These mAbs recognized Chlamydia -specific epitopes [ α 2→8-linked disaccharide of 3-deoxy- d - manno -octulosonic acid (Kdo) or the trisaccharide α Kdo-(2→8)-→Kdo] or those shared between chlamydial and Re-type LPS ( α Kdo, α →4-linked Kdo disacccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.     

Modulation of the antigenic phenotype of human breast carcinoma cells by modifiers of protein kinase C activity and recombinant human interferons

Summary In the present study we have analyzed the effect of a synthetic protein kinase C (PKC) activator 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol (ADMB) and the natural PKC-activating tumor-promoting agents 12-O-tetradecanoylphorbol 13-acetate (TPA) and mezerein on the antigenic phenotype of T47D human breast carcinoma cells. All three agents increased the surface expression of the tumor-associated antigen BCA 225 and various cellular antigens, including HLA class II antigens, intercellular adhesion molecule 1 (ICAM-1) and c-erbB-2. Expression of the same antigens was also upregulated to various extents in T47D cells by recombinant fibroblast (IFN) and immune (IFN) interferon. Shedding of BCA 225 from T47D cells was induced by TPA, mezerein, IFN and IFN, whereas ADMB did not display this activity. The ability of ADMB, TPA and mezerein to modulate the antigenic phenotype of T47D cells appears to involve a PKC-mediated pathway, since the PKC inhibitor, H-7, eliminates antigenic modulation. In contrast, the ability of IFN and IFN to enhance the synthesis, expression and shedding of BCA 225, as well as to enhance HLA class II antigens, c-erbB-2 and ICAM-1 expression, was either unchanged or modestly reduced by simultaneous exposure to H-7. Analysis of steady-state mRNA levels for HLA class I antigens, HLA class II-DR antigen, ICAM-1 and c-erbB-2 indicated that the ability of H-7 to inhibit expression of these antigens in TPA-, mezerein- and ADMB-treated cells was not a consequence of a reduction in the steady-state levels of mRNAs for these antigens. The results of the present investigation indicate that the biochemical pathways mediating enhanced antigenic expression in T47D cells induced by TPA, mezerein and the synthetic PKC activator ADMB are different from those induced by recombinant interferons. Furthermore, up-regulation of antigenic expression in T47D cells can occur by a PKC-dependent or a PKC-independent pathway.     

Selected low-cohesion variants of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus lack distinct antigens recognized by human antibodies

Actinobacillus actinomycetemconitans OMZ 346 A and Haemophilus aphrophilus OMZ 384 A, isolated on a synthetic selective and differentiating agar, show the highly cohesive and wall adherent growth in liquid medium which is typical for all primary oral isolates of these species. From each of them a low cohesion variant, OMZ 346 F and OMZ 384 F, respectively, was obtained by selection for cells growing in suspension. Screening of Western blots of these four strains with several human sera revealed the loss of a 4000 M_r antigen in both F strains. Human antibodies bound to the 400 M_r band material on preparative Western blots of the A strains were cluted with 4M magnesium chloride. These antibodies showed no cross-reaction between the 4000 M_r material of the two closely related species.     

Cell type specificity of a neural cell surface antigen recognized by the monoclonal antibody A2B5

Summary The monoclonal antibody A2B5 reacts with the surface membrane of most neurons in monolayer cultures of cerebellum, retina, spinal cord, and dorsal root ganglion of embryonic and early postnatal C57BL/6J mice maintained in vitro for culture periods of 2 to 10 days. A small percentage of astroglial cells also expresses A2B5 antigen in murine, chicken and rabbit cerebellum, in chicken retina, and in murine spinal cord and dorsal root ganglion. Less mature astroglial cells are stained for A2B5 antigen to a greater extent than the more mature astrocytes. Astrocytes from rat cerebellum and mouse retina were not found to express A2B5 antigen under the present culture conditions. Some of the less mature oligodendrocytes recognized by 04 antibodies express A2B5 antigen, while the more mature 01 antigen and galactocerebroside-positive oligodendrocytes were not found to be A2B5 antigen-positive. Fibroblasts or fibroblast-like cells do not express detectable levels of A2B5 antigen. After fixation of the cells with paraformaldehyde and ethanol, all cell types present in culture are labeled by the A2B5 antibody intracellularly.     

Identification of Toxoplasma gondii proteins binding human lactoferrin: a new aspect of rhoptry proteins function

In this paper, we report on the isolation, purification and identification of two Toxoplasma gondii membrane proteins binding human lactoferrin. Parasite membrane proteins were isolated using the commercial Mem-PER Eukaryotic Membrane Protein Extraction System. After purification by lactoferrin affinity chromatography, three protein bands were detected with the molecular mass of 74, 63 and 58 kDa, two of which (63 and 58 kDa) specifically bound biotin labeled human lactoferrin as examined by competitive inhibition. Further identification of latter proteins by ESI/MS/MS amino acid sequencing technique revealed those proteins as Toxoplasma ROP4 (band 63 kDa) and ROP2 (band 58 kDa) antigens known to be involved in many mechanisms essential for the parasite pathogenicity, including host lactoferrin acquisition as determined in this study.     

Identification of a novel adhesion molecule in human leukocytes by monoclonal antibody LB-2

Monoclonal antibody LB-2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus-immortalized normal B cells (EBV-B) and concanavalin A-stimulated blood mononuclear cells (Con A-BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester-induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV-B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.