乙型肝炎病毒表面抗原多肽图谱与亚型关系

用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western吸印法分析乙型肝炎病毒表面抗原(HBsAg)多肽组成,与蛋白标准品比较获得分子量23000～50000 8种多肽,用单一抗各亚型因子血清分析,证实a抗原决定簇存在于多种多肽中,d抗原决定簇主要由分子量23000的多肽组成,而y抗原决定簇则由分子量31000及34000的两种多肽组成。     

Role of antigen and,antibody in the regulation of the immune response

The enzyme dextranase could degrade antigenic dextran in vivo even when given 6-15 d after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 but not 48 h after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. To show whether antibody-mediated suppression of the immune response was determinant specific FITC-conjugated SRC were applied as immunogen and antibodies were raised both against the carrier (SRC) and the FITC hapten. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant-specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.     

Reduction and Reoxidation of Australia Antigen: Loss and Reconstitution of Particle Structure and Antigenicity

Reduction of Australia antigen with dithiothreitol resulted in the loss of antigenicity and morphological integrity. Oxidation of the reduced Australia antigen reconstituted both of these characters.     

Cryptodeterminant of a sperm maturation antigen on the mouse flagellar surface.

The determinant of a mouse sperm maturation antigen was examined morphologically and biochemically with monoclonal antibody T21 as a probe. The plasma membrane components of cauda epididymal spermatozoa were extracted with nonionic detergent Nonidet P-40 and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and by immunoblotting. Wheat germ agglutinin-lectin staining and immunoblotting indicated that the antigen recognized by T21 is a sialoglycoprotein of about 54,000 daltons (54 kDa). The antigenic determinant was more distinctly exposed after treatment with neuraminidase, as evaluated by immunohistochemistry, immunocytochemistry, and immunoblotting. The cryptic nature of the determinant was further confirmed by immunostaining nitrocellulose strips, subsequently digesting the strips with neuraminidase, and then reimmunostaining them. Results obtained by periodate oxidation treatment suggested that the epitope is a carbohydrate. Immunoperoxidase electron microscopy confirmed that the antigen is distributed on the flagellar plasma membrane of the sperm. This was demonstrated clearly when sperm were desialylated with neuraminidase. These results indicate that the 54 kDa sialoglycoprotein sperm maturation antigen has a cryptodeterminant which can be masked by a sialic acid residue, that is recognized by monoclonal antibody T21.     

Genetic regulation of the immune response to hepatitis B surface antigen (HBsAg). II. Qualitative characteristics of the humoral immune response to the a, d, and y determinants of HBsAg

It was previously demonstrated that the murine humoral immune responses to the common a and subtype-specific d determinants of HBsAg are H-2 restricted. The H-2q haplotype confers high responsiveness and the H-2s haplotype low responsiveness to nonresponsiveness to both determinants. We have now demonstrated that the H-2s haplotype also confers nonresponsiveness to the subtype-specific y determinant as well. Studies of H-2 congenic (nonresponder X responder)F1 and backcross mice indicated that responsiveness was inherited as a dominant trait, with no gene dosage effects observed. Qualitative characteristics of the humoral anti-a and anti-d responses were evaluated with respect to strain variation, kinetics, antigen specificity and antibody titer, affinity, and subclass distribution. Unique immune response patterns were observed for each H-2 haplotype studied. On the basis of these patterns, it was possible to construct a hierarchy of responsiveness to HBsAg of the ad subtype as follows: high responders, H-2q and H-2d; intermediate responders, H-2a greater than H-2b greater than H-2k; and nonresponders, H-2s.     

The demonstration of subtype (D or Y)-specific determinants on the surface of the presumed hepatitis B virus.

Dane particles isolated from the sera of HBsAg/ad and HBsAg/ay carriers were reacted with monospecific antibodies to the d and y subtype-specific determinants of HBsAg/. Dane particles from HBsAg/ad expressed the d determinant on their surfaces and those from HBsAg/ay sera contained the y specificity. Both complete (DNA-P and HBcAg) and defective (HBcAg alone) Dane particles expressed the subtype-specific determinants.     

Autoantibodies to the common antigenic determinant of group A streptococcal polysaccharide and epithelial cells of mammalian thymus and skin]

A cross-reactive (CR) antigenic determinant in the polysaccharide of group A streptococus and the mammalian thymus and skin was studied. The CR antigen was found in adult humans and human embryos irrespective of the blood group, as well as in the tissues of all the animal species studied, including rabbits immunized with streptococcus and producing antibodies to A polysaccharide. Evidence was obtained that the antibodies elaborated to the CR determinant of polysaccharide A and of the tissue epithelium should be classed as autoantibodies. It is suggested that reaction of these antobodies with the CR antigen in the thymus could serve as one of the causes of autoimmune thymitis during rheumatic fever.     

Role of the L3T4 antigen in T-cell activation. I. Description of a monoclonal IgM antibody to a distinct epitope (L3T4b) of the L3T4 antigen and its effect on interleukin 1-induced thymocyte proliferation

This report describes a new rat monoclonal IgM/k antibody, monoclonal antibody (MAb) 2B6, which reacts with a cell surface antigen present on a subpopulation of both thymocytes (85%) and peripheral T lymphocytes (55-60%). The antigen recognized by MAb 2B6 has multiple properties in common with the L3T4 antigen, as defined by the recently described MAb GK1.5. Thus, MAb 2B6 and MAb GK1.5 give very similar flow cytometry staining patterns on thymocytes, purified spleen T cells and all tested T-cell hybridomas. Depletion of MAb 2B6-positive cells with antibody and complement led to simultaneous depletion of MAb GK1.5-positive cells, and vice versa. Depletion of Lyt 2-positive cells led to enrichment of both MAb 2B6- and MAb GK1.5-positive cells. Both MAb 2B6 and MAb GK1.5 immunoprecipitate the same pattern of cell surface molecules from detergent extracts of radiolabeled thymocytes, the main components being a 55-kDa and a 115-kDa band. We therefore conclude that MAb 2B6 reacts with the L3T4 antigen. Interestingly, MAb 2B6 and MAb GK1.5 do not cross-block and therefore most probably react with distinct epitopes on the L3T4 molecule. The determinant recognized by MAb GK1.5 is called L3T4a. We suggest that the determinant recognized by MAb 2B6 be named L3T4b. As MAb 2B6 was selected for its ability to inhibit the action of interleukin 1 (IL-1) in the thymocyte costimulator assay, it is likely that the L3T4 molecule is functionally involved in the events taking place during IL-1 induction of thymocyte proliferation.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

Immunochemical studies of type IV and two group-like (Z) carbohydrate antigens of minute streptococci

Analysis of purified fractions of formamide extracts of Z₄IV and 8650 (Z₅) bacteria gave as composition rhamnose, glucose, galactose and N-acetyl-glucosamine in molar ratio’s for Z₄ antigen 9:0:2:5, for type IV antigen 4:4:4:1 and for Z₅ antigen 8:2:2:3. In contradistinction with other polysaccharide type antigens of minute streptococci all type IV reactivity was recovered from the buffer eluate of a DEAE cellulose column. The Z₅ antigen was present in both the water and the buffer eluate. Precipitin and inhibition reactions indicate that the serological reactions between both strains are cross reactions based on the presence of galactose in the determinant groups of type IV, group Z₄ and group Z₅ antigens. Inhibition reactions also suggest a role of β-galactosyl-glucose as immunodominant group of the Z₅ determinant. Partial acid hydrolysis of type IV antigen yielded four oligosaccharides. Analyses and inhibition reactions show that probably both trisaccharides β-galactosyl-glucosyl-galactose and β-galactosyl-glucosyl-rhamnose are determinant groups of the type IV antigen.     

Activation of Lyt-1+ and Lyt-2+ T cell cloned lines: stimulation of proliferation, lymphokine production, and self-destruction

Antigen-induced activation of a chicken gamma-globulin (CGG)-specific Lyt-1+ T cell clone measured both as a function of proliferation and immune interferon (IFN-gamma) production is restricted by a class II determinant of the major histocompatibility complex (MHC) mapped to the I-A subregion, as determined by studies with both recombinant inbred lines and monoclonal antibodies. Activation of Lyt-2+ picryl chloride (PC1)-specific cloned T cell lines by trinitrophenyl (TNP)-coupled spleen cells results in proliferation and the production of at least two lymphokines: lymphotoxin (LT) and IFN-gamma. This antigen-specific activation is restricted to a class I determinant of the MHC complex encoded in the K region. Thus, the common intracellular pathway leading to production of IFN-gamma by Lyt-1+ and Lyt-2+ T cells is mediated and restricted through different surface recognition units. The LT that is produced by antigen-specific activation of T cells not only kills fibroblasts, but it inhibits interleukin 2 (IL 2)-maintained T cells as well. Activation of T cells by concanavalin A (Con A) results in suicidal inhibition of proliferation and cell death by those clones that make LT, but not by those that produce only IFN-gamma under such induction conditions. These results indicate that it is neither Con A nor IFN-gamma that kills T cells, but LT. These results strongly suggest a self-regulatory role of LT in limiting continuing unrestricted T cell response to antigen activation.     

Monoclonal antibodies to O4 antigen of Vibrio parahaemolyticus

Four monoclonal antibodies were prepared against O4 antigen of Vibrio parahaemolyticus. All the antibodies were shown to be specific for O4 antigen by agglutination with heat-killed O-cells of the organism and precipitation with LPS preparations. The inhibition experiments of the precipitations with various sugars and oligosaccharides suggested that the combining sites of these hybridoma antibodies were directed to an antigenic determinant structure containing----3 and----6 linked D-glucose, D-galactose, and N-acetyl-D-galactosamine.     

Study of genetic control of synthesis of type III and 6th group-specific antigens of Flexner shigellae]

As revealed in experiments of interspecific and intraspecific crosses, the determinant responsible for the group-specific 6, as well as controling the synthesis of type III antigens of Sh. flexneri were localized near the lac-pro markers on the chromosome. In intraspecies crosses of bacteria of y variant with the donor Sh. flexneri 3c strain the percentage of linking of lac+a6+ was 32, and of lac+aIII+ --24. Genetic confirmation of the dependence of the function of the a6 (defined as att6) determinant on the presence of an antigenic complex 3,4 and of the association of the serological detection of the antigen III on the gene a6 was presented. On the basis of the data of Gemski et al it can be supposed that the determinant of the a6 served as the site of attachment of a specific converting phage.     

New Specificity of Australia Antigen

THE immunodiffusion laboratory at the Institute for Cancer Research frequently acts as a reference laboratory to test anti-Australia antigen sera for our colleagues in many parts of the world. Because Australia antigen is known to possess different antigenic specificities^1–4, a panel was established which consisted of Australia antigen specimens selected from hepatitis and Down's syndrome patients and from clinically normal residents of the Lau area in Malaita, British Solomon Islands. Sera from normal blood donors without Australia antigen were included as negative controls. All antisera received after August 1971 were tested against this panel to detect heterogeneity among both the antibodies tested and the antigens included in the panel. Immunodiffusion was performed in a seven-hole Ouchterlony pattern with the antiserum in the centre well and a positive Australia antigen control serum from a Pennsylvania Down's syndrome patient in the top and bottom wells. The patterns were cut in a layer of 1.1% agarose in veronal buffer, pH 8.2, on glass lantern slides^6,7.     

Identification and recovery of Leishmania antigen displayed on the surface membrane of mouse peritoneal macrophages infected in vitro

A murine monoclonal antibody (83L-2D3/a) to a dominant surface antigen of Leishmania braziliensis panamensis (WRAIR-470) recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages infected in vitro. This determinant, also demonstrable on the surface membrane of intracellular amastigotes, was not displayed by the macrophage until at least 6 hr post-infection. This delay in expression and the obvious negativity of all uninfected macrophages inherent to infected cultures implied that the leishmania determinant had an intracellular origin. Furthermore, expression was dependent upon maintenance of macrophage function. When the parasite burden became overwhelming, additional antigen processing ceased, and that which had accumulated was either shed into the medium or was internalized. Immunochemical analyses revealed that the 83L-2D3/a reactive epitope was part of a 15,000 dalton molecule, which in all likelihood represents a breakdown product of a major surface glycoconjugate that had been degraded in the phagolysosome.     

Identification of H-specific determinants in flagellin of four Escherichia coli strains

The H serogroup of Escherichia coli is determined by the flagellar antigen, flagellin. Sequence analysis of the flagellin gene, fliC, reveals a central variable region and the highly conserved N- and C-termini. This variable region has been shown to encode both H-specific and cross-reactive epitopes. Using polyclonal antibodies, we mapped the linear H-specific determinants in flagellin from four E. coli serotypes O157:H10, 0138:H14, O157:H42 and O157:H43. The specificity of all potential fragments was verified with 52 ECRC (Escherichia coli Reference Center) H-specific antisera. Our results indicated that: (a) a specific determinant of H10 flagellin (1263 bp long) maps to the region covering amino acid residues 305-331; (b) a specific determinant of H14 flagellin (1653 bp long) maps to the region covering amino acid residues 430-461; (c) a specific determinant of H42 flagellin (1281 bp long) maps to a region covering amino acid residues 171-201; and (d) a specific determinant of H43 flagellin (1506 bp long) maps to a region covering amino acid residues 200-260.     

Hopanoids of the methylotrophic bacteria Methylococcus capsulatus and Methylomonas sp. as possible precursors of C29 and C30 hopanoid chemical fossils

Abstract The majority of methicillin-resistant Staphylococcus aureus isolated in Australia since 1980 carries a determinant encoding resistance to a variety of nucleic acid-binding (NAB) compounds, including ethidium bromide (EB) acriflavine and propamidine isothionate. In addition, an EB-resistance determinant is frequently located on conjugative plasmids encoding aminoglycoside resistance. A comparison of these two determinants revealed that the EB-resistance determinant also encoded resistance to several NAB compounds. However, the NAB- and the EB-resistance determinants differed in both the number of NAB-compounds to which they expressed resistance, and the level of resistance expressed towards these compounds. Whilst these 2 determinants have several features in common, the results suggest that the NAB- and EB-resistance determinants are not identical.     

Prioritization of SNPs in y+LAT-1 culpable of Lysinuric protein intolerance and their mutational impacts using protein-protein docking and molecular dynamics simulation studies

Lysinuric protein intolerance (LPI) is a rare, yet inimical, genetic disorder characterized by the paucity of essential dibasic amino acids in the cells. Amino acid transporter y+LAT-1 interacts with 4F2 cell-surface antigen heavy chain to transport the required dibasic amino acids. Mutation in y+LAT-1 is rumored to cause LPI. However, the underlying pathological mechanism is unknown, and, in this analysis, we investigate the impact of point mutation in y+LAT-1's interaction with 4F2 cell-surface antigen heavy chain in causing LPI. Using an efficient and extensive computational pipeline, we have isolated M50K and L334R single-nucleotide polymorphisms to be the most deleterious mutations in y+LAT-1s. Docking of mutant y+LAT-1 with 4F2 cell-surface antigen heavy chain showed decreased interaction compared with native y+LAT-1. Further, molecular dynamic simulation analysis reveals that the protein molecules increase in size, become more flexible, and alter their secondary structure upon mutation. We believe that these conformational changes because of mutation could be the reason for decreased interaction with 4F2 cell-surface antigen heavy chain causing LPI. Our analysis gives pathological insights about LPI and helps researchers to better understand the disease mechanism and develop an effective treatment strategy.     

Allelic subtypic determinants of hepatitis B surface antigen (i and t) that are distinct from d/y or w/r.

A monoclonal antibody (I-18) was raised against an enneapeptide representing amino acids 125 to 133 of the product of the S gene of hepatitis B virus DNA [S(125-133) segment] with a sequence of Thr-Ile-126-Pro-Ala-Gln-Gly-Thr-Ser-Met. Another monoclonal antibody (T-7) was raised against an S(125-133) segment in which Ile-126 was replaced by Thr-126. In a panel of 16 samples of hepatitis B surface antigen (HBsAg) with known S gene sequences, I-18 reacted with 5 with Ile-126. T-7 reacted with 10 HBsAg samples with Thr-126; it did not, however, react with the remaining one of subtype ayw with Thr-126 flanked by Met-125 and Thr-127. The two allelic subtypic determinants, specified by Ile-126 and Thr-126 and distinct from d/y or w/r, were named i and t after isoleucine and threonine, which regulate them. They were expressed in a mutually exclusive fashion in 216 (83%) of 260 HBsAg samples from asymptomatic carriers. They were not detected in 36 (14%) samples; the failure to detect an i or t determinant was particularly common in HBsAg samples of subtype ayw (26 [79%] of 33). A part of the S gene sequence was determined for eight HBsAg samples without a detectable i or t determinant. They had an Ile-126 or Thr-126 residue that was flanked by Thr-127, not the Pro-127 commonly possessed by HBsAg samples displaying an i or t determinant. Expression of the i/t allele, therefore, would require Pro-127. In eight (3%) of the samples, both i and t determinants were detected; the presence of i and t on the selfsame HBsAg particles was verified by sandwiching the particles between I-18 and T-7. A point mutation from thymine to cytosine at nucleotide 377 in the S gene, contributing different second letters to codon 126 (ATT for Ile and ACT for Thr), would have been responsible for the assembly of HBsAg particles with both i and t determinants by means of phenotypic mixing.     

Antigen-specific CD4 effector T cells: Analysis of factors regulating clonal expansion and cytokine production: Clonal expansion and cytokine production by CD4 effector T cells

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4⁺ antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4⁺ T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRβ crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high “avidity” effector and memory T cells in response to pathogen are discussed.