Cell culture of mammalian thymic epithelial cells: growth, structural, and antigenic properties

The thymus plays an important role in the maturation and differentiation of T lymphocytes. Many of its functions have been attributed to its epithelial component. Past in vitro studies of putative thymic epithelial cells have been hampered by the inability to produce well-characterized cultures of these cells. Using lethally irradiated 3T3 cells as a feeder layer, we have succeeded in growing virtually pure cultures of thymic epithelial (TE) cells from rabbits, mice, and humans. Antikeratin staining provides an unambiguous criterion for positive identification of the epithelial cells. These cells were found to lack Ia-like or theta-like antigens. The ability to culture large quantities of mammalian TE cells should allow for their detailed functional characterization.     

The lipopolysaccharide of moraxella catarrhalis structural relationships and antigenic properties.

Moraxella catarrhalis has recently been shown to be both widespread and pathogenic, in contrast to previous reports. Several factors have been suggested as virulence factors, lipopolysaccharide (LPS) being one. Recent studies have shown the LPS to be without the O-chain, i.e. the polysaccharide part, and to have specific structural features corresponding to each of the three serogroups, A, B and C. The structures resemble in many respects those present in other Gram-negative nonenteric bacteria, with a galabiosyl element as a prominent common denominator. The presence of such common structures suggests that the LPS of these bacteria might be a part of a mechanism of survival for bacteria colonizing the human host.     

Protein polymorphism of a human plasma apolipoprotein D antigenic epitope

Based on our previous observation that monoclonal antibody anti-apoD-4E11 reacted with several HDL proteins we studied them further with three questions in mind: i) is there common protein polymorphism in healthy individuals? ii) how many proteins are present and what are their characteristics? iii) are they all apolipoproteins and do they have the same lipoprotein distribution as apoD? Isolated, delipidated apoD was used as a standard for radioimmunometric assay of plasma with antibody 4E11. The antigen varied from 3 to 11 mumol-equivalents of apoD per liter of plasma (equivalent to 5-20 mg apoD/dl plasma) with means of 6.1 and 6.8 mumol/l in men and women, respectively. Two-dimensional electrophoresis of plasma found up to eight 4E11-antigenic-proteins of different Mr, each heterogeneous in pI. All plasmas tested contained apoD and an Mr 38,000 antigen, the latter being the most immunoreactive. Six proteins of Mr 70,000-94,000 were found, but the number varied between subjects. Eighty nine percent of the plasma antigen was associated with lipoproteins: 83% with HDL and VHDL, 5% with LDL and VLDL. Lipoproteins of all sizes, separated by polyacrylamide gradient gel electrophoresis, contained the antigen. ApoD was almost the only 4E11-antigen in LDL, and was in two states: the one free, the other an apoD-apoB mixed disulfide complex. The apparent proportions of higher Mr antigens increased with increasing lipoprotein density, and the proportion of apoD decreased reciprocally. None of these 4E11-antigenic-proteins cross-reacted with antiserum to retinol-binding protein.     

Streptomycin-sensitivity in Streptomyces glaucescens is due to deletions comprising the structural gene coding for a specific phosphotransferase

Summary The wild type strain of Streptomyces glaucescens produces hydroxystreptomycin and has a natural resistance towards the streptomycin group aminoglycoside antibiotics. The inherent resistance is a genetically unstable character and mutant strains sensitive to streptomycins arise spontaneously at unusually high frequencies. The gene conferring streptomycin resistance was cloned and characterised as a streptomycin specific phosphotransferase. Hybridisation experiments show that the mutational event leading to sensitivity is due to large deletions, most likely on the chromosome, comprehending the structural gene coding for a streptomycin phosphotransferase and its flanking regions. Interspecific expression of the S. glaucescens phosphotransferase was found in Streptomyces lividans as well as in Escherichia coli.Abbreviations bp base pairs - EDTA ethylenediaminetetraacetic acid - kb kilobases' - TES n-tris(hydroxymethyl) methyl-2-aminoethane sulfonic acid     

Synthesis, photophysical effects, and DNA targeting properties of oxazole yellow-peptide bioconjugates

The design of a dsDNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency is described. The bioconjugate has the molecular recognition features of the polypeptide from a DNA-binding protein and the dsDNA-dependent fluorescence of an intercalating dye. The DNA sequence selectivity of the probe was characterized, as were the changes in photophysical properties of the dye upon covalent linkage to the peptide to assess whether such bioconjugates could function as molecular probes of gene sequences. The oxazole yellow-peptide bioconjugate exhibits DNA recognition and binding affinity comparable to the native Hin recombinase protein. Examination of photophysical effects to dye conjugation indicates a negligible affect on the fluorescence quantum yield. Fluorescence studies indicate this molecular probe is useful to determine the presence of a given DNA target sequence and gives negligible fluorescence in the absence of a given target site. Using the synthetic route described here, bioconjugates could be designed using different combinations of DNA recognition polypeptides and cyanine dyes to generate an array of sequence specific and wavelength specific probes.     

Mapping the antigenic epitope for a monoclonal antibody against lysozyme

A monoclonal antibody (HyHEL-5), prepared to chicken lysozyme c by the method of K?hler and Milstein, identified an antigenic site (epitope) that was shared by the lysozymes of seven different species of galliform birds. The lysozymes of two galliform species, bobwhite quail and chachalaca, shared only partial antigenic identity with the epitope defined by this antibody. Duck lysozyme did not react with the antibody at all. Amino acids that determined the epitope structure were tentatively identified by comparing the amino acid sequences of these lysozymes and assuming the antigenic changes produced by evolutionary substitutions are not due to long-range conformational changes. Arg 68 was identified as a determining amino acid. Arg 68 is hydrogen-bonded to Arg 45, and together these two amino acids form a basic cluster that may be a subsite of the epitope. The antibody inhibited lysis of Micrococcus lysodeikticus by chicken lysozyme. Additionally, Biebrich Scarlet, a dye that binds to the catalytic site, inhibited antibody binding to this lysozyme, which indicates that the epitope extends into the cleft region between Arg 45 and Arg 114. The epitope was hypothesized to involve a region measuring at least 13 x 6 x 15 A including the Arg 68-Arg 45 complex that borders the enzymatic catalytic site. Four other monoclonal antibodies to lysozyme have been partially characterized; each had a distinct pattern of binding specificity for various species of bird lysozymes.     

The porcine trophoblastic interferon-gamma, secreted by a polarized epithelium, has specific structural and biochemical properties.

At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-gamma), together with lesser amounts of IFN-delta, a unique species of type I IFN. This trophoblastic IFN-gamma (TrIFN-gamma) is an unprecedented example of IFN-gamma being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-gamma from other sources, either natural LeIFN-gamma (from adult leucocytes), or recombinant. Biologically active TrIFN-gamma is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-gamma which is formed of at least two polypeptide chains and four glycotypes. TrIFN-gamma collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N-acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l-fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-gamma molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-gamma on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-gamma a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium.     

Immunochemistry of scorpion toxins. Synthesis and antigenic properties of a model of a loop region specific to alpha-toxins

A region of the toxin II of the scorpion Androctonus australis Hector, possessing a loop structure, is shown to be antigenic. Some clear hints for the probable antigenic character of this region were obtained by the protruding properties of the loop region, as assessed by accessibility computations using atomic coordinates of the toxin and Lee-Richards algorithm. A synthetic replica of the loop region was obtained in a linear and cyclised form. Within the total anti-toxin antibody population, we have found and isolated those that recognize the model peptides. A high affinity binding of these specific antibodies to the parent toxin was demonstrated, affording experimental evidence for the antigenic properties of the loop region.     

Polypeptide conjugates comprising a beta-amyloid plaque-specific epitope as new vaccine structures against Alzheimer's disease

Immunotherapeutic approaches designed to induce a humoral immune response have recently been developed for possible vaccination to the treatment of Alzheimer's disease (AD). Based on the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, branched polypeptide conjugates with this epitope peptide were synthesized and characterized. In order to produce immunogenic constructs, the Abeta(4-10) epitope alone or together with a promiscuous T-helper cell epitope peptide (FFLLTRILTIPQSLD) were attached via thioether linkage to different branched chain polymeric polypeptides with Ser or Glu in the side chains. A single peptide containing both an Abeta(4-10) and T-helper cell epitope, joined by a dipeptide Cys-Acp spacer, was also attached through the thiol function to chloroacetylated poly[Lys(Seri-DL-Alax)] (SAK). Comparative binding studies of the conjugates with a monoclonal antibody against the beta-amyloid(1-17) peptide in mice were performed by direct ELISA. The conformational preferences of carriers and conjugates in water and in a 9:1 trifluoroethanol:water mixture (v/v) was analyzed by CD spectroscopy. Experimental data showed that the chemical nature of the carrier macromolecule, and the attachment site of the epitope to the carrier, have significant effects on antibody recognition, but have no marked influence on the solution conformation of the conjugates.     

Identification of an antigenic epitope and receptor binding domain of human C5a

Nine different murine anti-human C5a monoclonal antibodies have been produced and characterized. They exhibit Kas for the 125I-labeled ligand that range from 0.4 to 48 X 10(8) M-1, and they display limited cross-reactivity with C5a from other species. Each of these antibodies has been found to compete with the granulocyte C5a receptor for binding site(s) on the C5a polypeptide. Exploration of the antigenic topography of C5a revealed that the immunodominant portion of this glycopolypeptide resides between residues Lys20 and Arg37, with the area surrounding Cys27 being particularly important. In addition, a specific C5a derived tryptic peptide containing these amino acid residues competes with 125I-C5a for binding to the receptor. These observations are consistent with previously published data and suggest that this area of the C5a molecule is an important part of the receptor "recognition domain", and thus plays a critical role in the C5a receptor interaction.     

Immunogenic peptide comprising a mouse hepatitis virus A59 B-cell epitope and an influenza virus T-cell epitope protects against lethal infection.

The coronavirus spike protein S is responsible for important biological activities including virus neutralization by antibody, cell attachment, and cell fusion. Recently, we have elucidated the amino acid sequence of an S determinant common in murine coronaviruses (W. Luytjes, D. Geerts, W. Posthumus, R. Meloen, and W. Spaan, J. Virol. 63:1408-1412, 1989). A monoclonal antibody directed to this determinant (MAb 5B19.2) protected mice against acute fatal infection. In this study, BALB/c mice were immunized with a synthetic peptide of 13 amino acids corresponding to the binding site of MAb 5B19.2, which was either extended with an amino acid sequence of influenza virus hemagglutinin or conjugated to keyhole limpet hemocyanin. Both immunogens induced S-specific antibodies in mice, but only the hemagglutinin-peptide construct protected them against lethal challenge. In contrast to mouse hepatitis virus type 4 (MHV-4), MHV-A59 was not neutralized in vitro by MAb 5B19.2. Neither MHV-A59 nor MHV-4 was neutralized in vitro by antibodies comprising by the synthetic peptides. Our results demonstrated that antibodies elicited with a synthetic peptide comprising a B-cell epitope and a T-helper cell determinant can protect mice against an acute fetal mouse hepatitis virus infection.     

Design, development and synthesis of mixed bioconjugates of piperic acid-glycine, curcumin-glycine/alanine and curcumin-glycine-piperic acid and their antibacterial and antifungal properties

In the present communication different curcumin bioconjugates viz. 4,4'-di-O-glycinoyl-curcumin, 4,4'-di-O-d-alaninoyl-curcumin, 4,4'-di-O-(glycinoyl-di-N-piperoyl)-curcumin, 4,4'-di-O-piperoyl curcumin, curcumin-4,4'-di-O-beta-d-glucopyranoside, 4,4'-di-O-acetyl-curcumin along with piperoyl glycine, have been synthesised and characterised by spectra UV, (1)H NMR and elemental analysis. All the covalent bonds used are biodegradable. This makes these derivatives as potent prodrugs, which can get hydrolysed at the target sites. These bioconjugates were tested in vitro against different bacteria and fungi. The 4,4'-di-O-(glycinoyl-di-N-piperoyl)-curcumin and 4,4'-di-O-acetyl-curcumin are more effective than Cefepime, an antibacterial drug available in market, at the same concentration. The 4,4'-di-O-(glycinoyl-di-N-piperoyl)-curcumin and 4,4'-di-O-piperoyl curcumin had antifungal activity in vitro almost comparable with fluconazole, the most popular antifungal drug. The enhanced activity of these bioconjugates vis-a-vis the parent molecule that is curcumin may be due to improved cellular uptake or reduced metabolism of these bioconjugates resulting in building up of enough concentration inside the infected cells. It opens a new era for exploring suitably designed curcumin bioconjugates as potential antibacterial/antifungal drugs.     

An in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context

Presently X-ray crystallography of protein-antibody complexes is still the most direct way of identifying B-cell epitopes. The objective of this study was to assess the potential of a computer-based epitope mapping tool (EMT) using antigenic amino acid motifs as a fast alternative in a number of applications not requiring detailed information, e.g. development of pharmaceutical proteins, vaccines and industrial enzymes. Using Gal d 4 as a model protein, the EMT was capable of identifying, in the context of the folded protein, amino acid positions known to be involved in antibody binding. The high sensitivity and positive predictive value of the EMT as well as the relevance of the structural associations suggested by the EMT indicated the existence of amino acid motifs that are likely to be involved in antigenic determinants. In addition, differential mapping revealed that sensitivity and positive predictive value were dependent on the minimum relative surface accessibility (RSA) of the amino acids included in the mapping, demonstrating that the EMTs accommodated for the fact that epitopes are three-dimensional entities with various degrees of accessibility. The comparison with existing prediction scales demonstrated the superiority of the EMT with respect to physico-chemical scales. The mapping tool also performed better than the available structural scales, but the significance of the differences remains to be established. Thus, the EMT has the potential of becoming a fast and simple alternative to X-ray crystallography for predicting structural antigenic determinants, if detailed epitope information is not required.     

Elucidation of structural and antigenic properties of pneumococcal serotype 11A, 11B, 11C, and 11F polysaccharide capsules

Despite the emerging impact of serogroup 11 serotypes in Streptococcus pneumoniae epidemiology, the structures of serogroup 11 capsule types have not been fully elucidated, particularly the locations of O-acetyl substitutions. Here, we report the complete structures of the serotype 11B, 11C, and 11F polysaccharides and a revision to the serotype 11A capsular polysaccharide using nuclear magnetic resonance (NMR). All structures shared a linear, tetrasaccharide backbone with a pendant phosphopolyalcohol. Three of four saccharides are conserved in all serotypes. The individual serotype capsules differed in the identity of one saccharide, the pendant phosphopolyalcohol, and the O-acetylation pattern. Though the assigned locations of O-acetate substitutions in this study differed from those of previous reports, our findings were corroborated with strong correlations to serology and genetics. We examined the binding of serotyping sera to serogroup 11 polysaccharides by using flow cytometry and an inhibition-type enzyme-linked immunosorbent assay (ELISA) and found that de-O-acetylation of capsular polysaccharides by mild hydrolysis decreases its immunoreactivity, supporting the crucial role of O-acetylation in the antigenicity of these polysaccharides. Due to strong correlations between polysaccharide structures and capsule biosynthesis genes, we were able to assign target substrates for the O-acetyltransferases encoded by wcwC, wcwR, wcwT, and wcjE. We identified antigenic determinants for serogroup 11 serotyping sera and highlight the idea that conventional serotyping methods are not capable of recognizing all putative variants of S. pneumoniae serogroup 11.     

Functional, structural, and antigenic similarities of guinea pig anti-phosphorylcholine antibodies.

The humoral immune response to PC was measured in guinea pigs. PC-vaccine stimulated IgM and IgG2, but little IgG1, anti-PC -antibodies. No memory was induced and immunization in CFA produced tolerance. PC-KLH, on the other hand, stimulated IgM, IgG2, and IgG1 anti-PC antibodies with carrier-specific memory. Hapten inhibition of plaque formation showed uniform binding patterns with minor, but significant, differences between antiPC-vaccine and anti-PC-KLH antibodies. The antibodies were characterized by IEF and idiotypic analyses. Early after immunization with PC-vaccine, guinea pigs had restricted IEF patterns which in inbred, but not outbred animals were indistinguishable between individuals. These patterns remained restricted but more individualized with time after immunization. Anti-PC-KLH antibodies showed more heterogeneity and individuality. However, these structurally heterogeneous antibodies reacted equivalently with rabbit anti-idiotype antisera and therefore must share common structural features, regardless of isotype or the genetic background of the guinea pig.     

Synthesis and antigenic properties of reduced peptide bond analogues of an immunodominant epitope of the melanoma MART-1 protein.

Backbone modifications have been introduced into the melanoma derived peptide MART-1(27-35) to increase its binding to class I major histocompatibility complex HLA-A2 molecule, and ultimately to enhance its immunogenicity. Each analogue was obtained by replacing one peptide bond at a time in the natural epitope by the aminomethylene (CH2-NH) surrogate. All analogues displayed an increased resistance to proteolysis. Interestingly, the comparative results showed that five analogues bound more efficiently to HLA-A2 than the parent peptide. On the other hand, two pseudopeptide/HLA-A2 complexes were recognized by one melanoma-specific T cell clone. Close examination of the impact of such modifications at the molecular level provides useful supports for the rational design of stable compounds with applications in anti-tumour specific immunotherapy and in vaccine development.     

Light-responsive bioconjugates as novel tools for specific capture of biologicals by photoaffinity precipitation

Light-responsive bioconjugates are synthesized by a two-step protocol calling first for cotelomerization (chain-transfer polymerization) of N-isopropylacrylamide and N-acryloxysuccinimide. The desired bioligand (biotin) is used in modified form as chain-transfer agent in this step. As a consequence, 100% of the produced bioconjugates carry this group. In a second step, the cotelomers (bioconjugates) are rendered photoresponsive by linking a chromophore ((3-aminopropyloxy)azobenzene) group to the N-acryloxysuccinimide side chains. The resulting structures show a critical solution temperature in pure water of 16 degrees C when the azo groups in the side chains are predominately in the (stable) trans state. Irradiation with UV light (330 nm) switches the azo group into the more hydrophilic cis state, and the critical solution temperature rises to 18 degrees C. Irradiation with visible light (> 440 nm) switches the group back to the trans state. Adjusting the temperature to an intermediate level, the bioconjugates are used to demonstrate the concept of photo affinity precipitation, i.e., the specific capture and recovery by light-induced precipitation of a target molecule (avidin) from a serum-containing cell-culture supernatant. The avidin was obtained in highly purified form; no nonspecific copurification of protein impurities was observable.     

Localization and characterization of a specific linear epitope of the Brucella DnaK protein

We have previously produced and characterized four monoclonal antibodies to the Brucella DnaK protein which were derived from mice infected with B. melitensis or immunized with the B. melitensis cell wall fraction. By use of a recombinant DNA technique, we have localized a linear epitope, recognized by two of these monoclonal antibodies (V78/07B01/G11 and V78/09D04/D08), in the last 21 amino acids of the C-terminal region of the Brucella DnaK protein. The C-terminal region has been reported to be the most variable region among DnaK proteins. The two other monoclonal antibodies (A53/09G03/D02 and A53/01C10/A10) failed to react with the recombinant clones and might recognize discontinuous epitopes of the Brucella DnaK protein. The four monoclonal antibodies reacted with all recognized Brucella species and biovars in immunoblotting after SDS-PAGE. Monoclonal antibodies V78/07B01/G11 and V78/09D04/D08 did not react with reported cross-reacting bacteria nor with bacteria of the α-2 subdivision of the class Proteobacteria for which a close genetic relationship with Brucella spp. has been reported. However, monoclonal antibodies A53/09G03/D02 and A53/01C10/A10 reacted with Phyllobacterium rubiacearum and/or Ochrobactrum anthropi, both bacteria of the α-2 subdivision of the class Proteobacteria. The Brucella genus DnaK specific epitopes could be of importance for diagnostic purposes.