Immune response potential to poly(Tyr,Glu)-poly(DLAla)--poly(Lys) of human T cells of different donors.

Human peripheral blood T cells of normal donors were activated in vitro with autologous adherent cells pulsed with poly(LTyr,LGlu)-poly(DLAla)--poly(LLys) [abbreviated (T,G)-A--L]. The "educated" T cells were tested: (i) for their ability to produce a (T,G)-A--L-specific T cell-replacing factor in the cooperation with B cells for antibody responses in vivo or in vitro and (ii) for their ability to proliferate in the presence of a second stimulus of (T,G)-A--L. Results of screening of 66 donors demonstrated that educated T cells of about 50% of the donors produced an active (T,G)-A--L-specific factor, whereas activated cells of only half of the factor producers were capable of proliferating in the presence of the antigen. Thus, as reported for all other species studied, human individuals differ in their response potential to (T,G)-A--L.     

Efficient genetically controlled formation of antibody to a synthetic antigen [poly(LTyr, LGlu)-poly(DLAla)- -poly(LLys)] covalently bound to a synthetic adjuvant (N-acetylmuramyl-L-alanyl-D-isoglutamine).

The synthetic polypeptide antigen poly(LTyr, LGlu)-poly(DLAl)- -poly(LLys)[T,G)-A- -L] was covalently linked to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal adjuvant-active structure that can substitute for Mycobacteria in complete Freund's adjuvant. When injected in aqueous solution into mice, the completely synthetic conjugate elicited significant antibody responses specific to (T,G)-A- -L, whereas (T,G,)-A- -L alone administered under the same conditions did not lead to antibody production. The conjugate was much more efficient in eliciting (T,G)-A- -L responses than was a mixture of DMP and (T,G)-A- -L. One hundred micrograms of MDP mixed with 10 micrograms of (T,G)-A- -L resulted in production of (T,g)-A- -L-specific antibodies. However, the titers obtained were much lower than those observed with 10 micrograms of the conjugate, MDP-(T,G)-A- -L, which contained less than 1 microgram of MDP. MDP was enhanced when the mixture was administered in incomplete Freund's adjuvant, the adjuvant did not significantly affect the (T,G)-A- -L-specific antibody responses in mice immunized with MDP-(T,G)-A- -L. The isoelectric focusing pattern of antibodies obtained with MDP-(T,G)-A- -L was similar to that obtained after immunization with (T,G)-A- -L in complete Freund's adjuvant. The pattern of high-responder and low-responder mice to (T,G)-A- -L, the immune response to which is genetically controlled, was retained when MDP-(T,G)-A- -L was used as the immunogen. Conjugation of (T,G)-A- -L was creased the immunogenicity of MDP and affected its biological properties. It is thus possible to obtain efficient immune responses to synthetic polypeptide antigens that produce poor reactions when injected in aqueous solution by conjugating them to small molecular weight synthetic adjuvants.     

Change in specificity of antibodies to a random synthetic branched polypeptide in mice tolerant to its ordered analogs

The crossreactivity between the random synthetic polypeptide antigen, (Tyr,Glu)-poly(DLAla)- -poly(Lys), and its ordered sequence analogs, (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys) and (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys), has been studied on the level of tolerance induction. Induction of tolerance to the random (Tyr,Glu)-poly(DLAla)- -poly(Lys) affected the response of the tolerant mice to the homologous antigen as well as to (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys), which was shown previously to represent the major determinant of (Tyr,Glu)-poly(DLAla)- -poly(Lys). In contrast, these mice responded with high antibody titers to the hardly crossreacting (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys). Mice tolerant to the ordered peptide antigen (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys) did not respond to the homologous polypeptide; however, their immune response to either (Tyr-Glu)-poly(DLAla)- -poly(Lys) or (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys) was not affected. Mice that were tolerant to (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(Lys) responded well to (Tyr-Glu-Tyr-Glu)-poly(DLAla)- -poly(Lys). Furthermore, these mice produced high antibody titers after immunization with the random (Tyr,Glu)-poly(DLAla)- -poly(Lys). However, the antibodies produced were not specific to the major determinant of (Tyr,Glu)-poly(DLAla)- -poly(Lys), namely, Tyr-Tyr-Glu-Glu, but were directed to minor determinants of the random polypeptide, including Tyr-Glu-Tyr-Glu, which are not immunopotent when nontolerant mice are immunized with (Tyr,Glu)-poly(DLAla)- -poly(Lys). Thus, whereas antigenic specificity reflects itself also at the level of tolerance induction, the animals that had been made tolerant are capable of responding to previously silent antigenic determinants.     

Cellular analysis of specificity of antibodies and of delayed type hypersensitivity responses toward some structurally related synthetic antigens: Boosting is determined by specificity of T cells

The crossreactivity between the random synthetic polypeptide antigen poly(Tyr,Glu)-poly(DLAla)--poly(Lys) [(T,G)-A--L] and its ordered-sequence analogs (Tyr-Tyr-Glu-Glu)-poly(DLAla)--poly(Lys) [(T-T-G-G)-A--L] and (Tyr-Glu-Tyr-Glu)-poly(DLAla)--poly(Lys) [(T-G-T-G)-A--L] at the level of humoral and cellular responses was studied. For delayed type hypersensitivity responses, (T,G)-A--L-activated T cells could be challenged with the homologous antigen as well as with the ordered analogs. T cells activated by (T-T-G-G)-A--L could be challenged with either the homologous antigen or with (T,G)-A--L but not with (T-G-T-G)-A--L. Similarly, no cross stimulation was observed between (T-G-T-G)-A--L-activated cells and (T-T-G-G)-A--L, whereas (T,G)-A--L could challenge the latter cells to mediate significant responses. Similar but not identical cross reactions were observed when primed spleen cells or lymph nodes were transferred to irradiated recipients that were boosted for the production of antibodies. In contrast to observations at the level of cellular responses, (T-G-T-G)-A--L-primed spleen or lymph node cells could not be boosted with (T,G)-A--L for the production of detectable amounts of antibodies, although boosting with the homologous antigen resulted in significant levels of (T-G-T-G)-A--L-specific antibodies. Transfer experiments in which mixtures of T and B cells, each primed to a different ordered polypeptide antigen, were injected into irradiated recipients showed that successful cooperation occurs provided that the boost is given with the T-cell-specific antigen. The antibodies produced were specific to the antigen used for B-cell priming. The T-cell-B-cell collaboration probably occurs through specific determinants that are shared between the two antigens in which the ordered peptides are attached to the same multichain polymer and that are recognized by both the T and the B cells.     

Functional helper activity of monoclonal T cell populations: antigen-specific and H-2 restricted cloned T cells provide help for in vitro antibody responses to trinitrophenyl-poly(LTyr,Glu)-poly(DLAla)--poly(LLys).

The ability of long-term cultured and monoclonal T cell populations to provide antigen-specific help was assessed in a system of Ir gene-controlled in vitro antibody responses to soluble antigens. T-cell colonies and monoclonal T-cell lines were generated which proliferated specifically in response to poly(LTyr,Glu)-poly(DLAla)--poly(LLys) [(T,G)-A--L] and were I-A restricted in these proliferative responses. These (T,G)-A--L-specific T-cell populations were evaluated for their ability to help unprimed and T-cell depleted spleen cell populations in the generation of antibody responses to trinitrophenyl (TNP)-(T,G)-A--L in vitro. It was found that long-term T-cell lines, including monoclonal T-cell populations derived by limiting dilution, were highly efficient helper cells for IgM responses to TNP-(T,G)-A--L. These helper T cells were both antigen-specific and I-A restricted in their ability to be activated and to cooperate with T-cell depleted spleen cell populations. Once specifically activated, however, these clones provided help that was antigen nonspecific. These studies have thus demonstrated the ability of antigen-specific and H-2-restricted monoclonal T-cell populations to provide help for responses to soluble antigens in vitro.     

Idiotypic analysis of monoclonal antibodies to poly(Glu60Ala30Tyr10).

Fifteen hybridoma anti-poly(Glu60Ala30Tyr10) (anti-GAT) antibodies were analyzed for the presence of a common set of idiotypic specificities associated with murine anti-GAT antibodies, termed CGAT idiotype, which are present on the anti-GAT antibodies of all mouse strains. Thirteen of these monoclonal anti-GAT antibodies expressed a major fraction of CGAT idiotypic specificities. However, the remaining fraction of CGAT idiotypic specificities were not detected in individual or pooled hybridoma anti-GAT antibodies. Anti-idiotypic antisera made against each of the 15 hybridoma anti-GAT antibodies preferentially bound homologous ligand and showed minimal binding activity to specifically purified serum anti-GAT antibodies. Furthermore, the diversity of the hybridoma anti-GAT antibodies was demonstrated by the presence of individual idiotypic specificities on each of the hybridoma anti-GAT antibodies. However, relatedness among some of the hybridoma antibodies was also apparent since idiotypic analysis revealed that some hybridoma anti-GAT antibodies shared cross-reactive idiotypic specificities not associated with CGAT idiotype. The genetic mechanisms which could account for the generation of such antibody diversity are discussed.     

Inhibition of dual Ir gene-controlled T-lymphocyte proliferative response to poly (Glu56Lys35Phe9)n with anti-Ia antisera directed against products of either I-A or I-C subregion.

Previous studies have demonstrated that both the antibody and T-lymphocyte proliferative immune responses to poly(Glu53Lys36Phe11)n (GLphi) are under the control of two major histocompatibility-linked immune response (Ir) genes. One gene, termed Ir-GL phi-alpha, has been mapped to the I-C or I-E subregion of the major histocompatibility complex, while the other, termed Ir-GL phi-beta, has been mapped to the I-A subregion. In this paper we examine the effect of anti-I-region-associated (Ia) antisera on the T-lyphocyte proliferative response to GL phi. Antibodies directed against Ia antigens coded for by genes in either the I-A or I-C subregion were found to inhibit the proliferative response to GLphi. These results suggest that a function mediated by two Ir gene products can be blocked by anit-Ia antisera directed against either one, and thus, that both products are expressed on the cell surface.     

Role of antigenic structure in cell to cell cooperation.

Two synthetic polypeptides which differ only in the order of amino acids in their NH2-terminal side chains, namely, (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(LLys) and (Tyr-Glu-Try-Glu)-poly(DLAla)- -poly(LLys), were found to be under different genetic control. By three different in vivo systems for thymus-derived cell depletion, it was demonstrated that (Tyr-Tyr-Glu-Glu)-poly(DLAla)- -poly(LLys), which represents the random poly(Tyr,Glu)-poly(DLAla)- -poly(Lys) in the pattern of immune responses and in the quality of antibodies they elicit, is thymus-dependent whereas (Tyr-Glu-Tyr-Glu)-poly(DLAla)-poly(LLys) does not require thymus-derived cell help for efficient antibody production. Therefore, the two ordered polypeptides which are similar chemically differ in parameters, not yet determined, which affect their capability to trigger bone marrow-derived cells.     

Recombinant proapoA-I(Lys107del) shows impaired lipid binding associated with reduced binding to plasma high density lipoprotein.

In the present study apoA-I (Lys 107del), a naturally occurring human apoA-I variant with a deletion of Lys 107, was expressed in E. coli to examine the effect of this mutation on lipid binding, cholesterol efflux and lecithin:cholesterol acyltranferase (LCAT) activation. Dimyristoyl phosphatidylcholine (DMPC) binding studies revealed slow interaction of proapoA-I(Lys107del) with DMPC relative to normal proapoA-I. After preincubation with human plasma lipoprotein (d<1.225 g/ml) for 1 h at 37 degrees C, 125I-labeled normal proapoA-I chromatographed as a single peak with the high density lipoprotein (HDL) fraction, whereas 125I-labeled proapoA-I(Lys107del) chromatographed with both HDL and free proapoA-I (26% of the radioactivity). Circular dichroism measurements showed that the alpha-helical content of lipid-bound proapoA-I (Lys107del) was reduced to 64 versus 73% of normal proapoA-I. Non-denaturing gradient gel electrophoresis of reconstituted HDL assembled with either proapoA-I(Lys107del) or normal proapoA-I showed that the mutation led to the formation of a second population of smaller rHDL particles. DMPC/proapoA-I(Lys107del) and normal DMPC/proapoA-I complexes exhibited a similar capacity to promote cholesterol efflux from fibroblasts. ProapoA-I (Lys107del) also activated LCAT similar to wild type proapoA-I and human plasma apoA-I. We conclude that deletion of Lys 107 substantially alters the lipid binding properties of the protein, which correlated with reduced binding to plasma HDL in vitro, but did not affect the capacity of the mutant/lipid complex to promote cholesterol efflux or activate LCAT.     

Fine specificity of antibodies to poly(Glu60Ala30Tyr10) produced by hybrid cell lines.

The polyethylene glycol-mediated cell fusion technique has been used to analyze the diversity of the antibody response to the terpolymer poly(Glu60Ala30Tyr10)(GAT). Nine stable clones (all producing IgM K anti-GAT antibodies) were isolated from a fusion between P3-X63-Ag8 myeloma cells and spleen cells from a DBA/2 mouse sensitized to GAT 5 days earlier. Seven other clones (producing IgG K anti-GAT antibodies) were derived from another fusion between NSI myeloma cells and spleen cells of (C57BL/6 X DBA/2)F1 hybrid mice hyperimmunized with GAT. These 16 anti-GAT antibodies were grouped according to their pattern of reactivity with GAT and the two related polymers of poly(Glu60Ala40) (GA) and poly(GLU50Tyr50) (GT). Two monoclonal anti-GAT antibodies (IgM F9-102.2 and IgG F17-148.3) demonstrated crossreactivity with GA but failed to crossreact with GT determinants. In contrast, the remaining 14 hybridoma antibodies demonstrated preferential reactivity with GAT but also exhibited crossreactive binding to GT and in some cases GA. There was a correlation between the fine specificity pattern and the presence of a common anti-GAT idiotype on these antibodies. Thus, the hybridoma anti-GAT antibodies which reacted with GT shared crossreactive idiotypic determinants (CGAT) present in mouse anti-GAT immune sera. In contrast, the monoclonal F9-102.2 and F17-148.3 antibodies that failed to bind to GT lacked the major CGAT idiotypic determinants.     

Structural bases for public idiotypic specificities of monoclonal antibodies directed against poly(Glu60Ala30Tyr10) and poly(Glu60Ala40) random copolymers.

NH2-terminal amino acid sequences of heavy and light chains of seven poly(Glu60Ala30Tyr10) (GAT) specific hybridoma products derived from DBA/2 and (DBA/2 X BALB/c)F1 hybrid mice and those of BALB/B polyclonal antibodies have been determined over the first 40 residues. Comparison of these sequences with those of nine other GAT or poly(Glu60Ala40) (GA) specific hybridoma products previously reported allowed the following conclusions. (i) Sequences of hybridoma H and L chains are present in the pool of polyclonal antibodies. (ii) The public CGAT (or pGAT) idiotypic specificities are strictly confined to antibodies exhibiting limited heterogeneity with regard to both the variable heavy (VH) and the variable kappa (V kappa) sequences that may be accounted for by one and two germ-line genes, respectively. (iii) The public idiotypic specificities GA-1, expressed by some anti-GAT and most anti-GA antibodies, make use of the same (or similar) VH germ-line genes as the CGAT or pGAT antibodies but possess a distinctive V kappa sequence. (iv) Antibodies expressing neither of the alternative public specificities mentioned above appear to be more heterogeneous and express VH and V kappa sequences that were found to differ from the basic structures defining the CGAT (pGAT) or GA-1 correlates. It is concluded that CGAT (or pGAT) and GA-1 public idiotypic specificities are germ-line markers of both VH and V kappa regions, an observation in agreement with previously reported serological data.     

Genetic control of major histocompatibility complex-linked immune responses to synthetic polypeptides in man: poly(L-phenylalanine, L-glutamic acid)-poly (DL-alanine)--poly(L-lysine) and L-glutamic acid, L-alanine, L-tyrosine (60:30:10).

Vigorous lymphoproliferative responses to synthetic polypeptides poly(L-phenylalanine, L-glutamic acid)-poly(DL-alanine)--poly(L-lysine) [( Phe,G)-A--L], and L-glutamic acid, L-alanine, L-tyrosine (60:30:10) (GAT) were observed in cells from 92 unrelated subjects. Thirty-three percent responded to (Phe,G)-A--L and 77% to GAT. No HLA association was observed with responses to these two antigens. Family studies indicated that two complementary immune response (Ir) genes are required for response to each antigen. Eleven matings were informative for linkage analysis between HLA and these Ir genes. Families in which the complementary genes are in coupling gave maximal lod scores (log of the odds) of 4.50 for (Phe,G)-A--L and 7.57 for GAT for 0 = 0. In a HLA-B/D recombinant family, the Ir- PheGAL genes are mapped towards the HLA-D region. The localization of Ir-GAT genes close to HLA-B was provided by a HLA-A/B recombinant.     

Turning (Ir gene) low responders into high responders by antibody manipulation of the developing immune system.

The ability of helper T cells directed against trinitrophenyl-modified syngeneic spleen cells to recognize low-hapten densities on target cells is under major histocompatibility complex-linked Ir gene control. Thus, BALB/c (H-2d) mice are low responders while H-2 congenic BALB.C3H (H-2k) mice are high responders. Immunization of adult BALB/c mice with the monoclonal antibody F6(51), directed to shared idiotopes by anti-trinitrophenyl antibodies and clonal receptors on anti-trinitrophenyl-self helper T cells, leads to the production of high titers of circulating idiotype, has no influence on helper T cell idiotypic profiles, but shifts to a high-responder phenotype the ability of helper T cells to recognize low-hapten densities. These effects on Ir gene phenotype are even more striking in untreated progenies from F6(51)-immunized BALB/c females, which are better responders than genetically high-responder BALB.C3H mice, although completely different in the expression of the F6(51)-defined clonotype. The general significance of these findings on Ir gene-directed T-cell repertoire selection is discussed, for they constitute formal evidence against antigen-presentation as a mechanism of Ir gene effects and strong support for the importance of maternal influences on the development of T-cell repertoires.     

Antigenicity of putative phospholipid membrane-binding residues in factor VIII

Most inhibitory antibodies to human factor VIII (fVIII) bind toepitopes in the A2, ap-A3, or C2 domains. The anticoagulant action of antibodies to the C2 domain is due to inhibition of bindingof fVIII to phospholipid. The x-ray structure of the human fVIII C2domain shows a putative hydrophobic, 3-prong, phospholipid membrane-binding site consisting of Met2199/Phe2200, Val2223, andLeu2251/Leu2252. Additionally, Lys2227, near Val2223, is part ofa ring of positively charged residues that may contribute to electrostatic interaction of fVIII with negatively chargedphosphatidylserine. In this study, 8 active mutants of human fVIII(Met2199Ile, Leu2252Phe, Phe2200Leu, Val2223Ala, Lys2227Glu,Met2199Ile/Phe2200Leu, Val2223Ala/Lys2227Glu, andMet2199Ile/Phe2200Leu/Val2223Ala/Lys2227Glu), which were constructed onthe basis of differences between human, porcine, murine, and caninefVIII at proposed phospholipid binding sites, were expressed. Theantigenicity of the mutants toward 5 C2-specific polyclonal humanantibodies was measured by using the Bethesda assay. A human monoclonalanti-C2 antibody, BO2C11, and a murine C2-specific monoclonal antibody,NMC VIII-5, were also included in the analysis. In comparison withwild-type, B-domainless fVIII, the Met2199Ile, Phe2200Leu, and Leu2252single mutants had lower antigenicity toward most of the inhibitors. Incontrast, the Val2223Ala and Lys2227Glu mutants usually showedincreased antigenicity. These results suggest that C2 inhibitorsfrequently target the Met2199/Phe2200 and Leu2251/Leu2252 -hairpinsand are consistent with the hypothesis that these residues participatein binding to phospholipid membranes. In contrast, Val2223 and Lys2227may oppose antibody binding sterically or through stabilization of alow-affinity membrane-binding conformation of the C2 domain.     

Idiotypic analysis of antibodies to poly(Glu60Ala30Tyr10): interstrain and interspecies idiotypic crossreactions.

A common idiotype was defined by a guinea pig anti-idiotypic antiserum made against D1.LP antibodies specific to the synthetic terpolymer poly(Glu60Ala30Tyr10), referred to as GAT. This idiotype was found in the anti-GAT antibodies of all individuals of all inbred strains of mice tested. Due to its common (C) occurrence in the anti-GAT antibodies of all mouse strains, it was termed the CGAT idiotype. The presence of CGAT idiotype in mice is independent of the expression of various Ig-1, H-2, or Vg genetic markers. In contrast, antibodies produced in response to the crossreactive poly(Glu60-Ala40), termed GA, bound to the GAT ligand but did not bear the CGAT idiotype. This suggests that GAT, although bearing GA determinants, preferentially stimulates clones of CGAT idiotype. Some of the CGAT idiotypic specificities were also identified in sera of WF inbred rats after immunization with GAT. However, no significant levels of CGAT idiotype were detected in LEW rats, F344 rats, guinea pigs, or rabbits, although measurable quantities of anti-GAT antibody were present.     

Localization, synthesis, and activity of an antigenic site on influenza virus hemagglutinin.

This paper reports the antigenicity of the fusion region of the influenza virus hemagglutinin (HA). Two peptides, comprising the fusion region (residues 1-11 of the HA2 part of HA) of strain A and strain B influenza virus, were synthesized and their abilities to bind rabbit, goat, and human anti-influenza antibodies were determined. In addition, 30 anti-HA monoclonal antibodies were examined for their ability to bind the synthetic peptides. In quantitative immunoadsorbent titrations, the two peptides bound considerable amounts of antibodies in rabbit and goat antisera against virus or HA of the A or B strain as well as in several human sera from patients recovering from influenza A. Of the 30 anti-HA monoclonal antibodies, 5 bound completely and 4 bound partially to the peptides. Antibodies were raised in rabbits against the peptides by immunizing with peptide-bovine serum albumin conjugates or with the free peptides. Anti-peptide antibodies were bound by HA and by the intact virus of the respective strain. However, these antisera failed to exhibit significant virus neutralizing activity. In contrast, the monoclonal antibodies that reacted with these peptides inhibited viral infectivity. The results clearly show that residues 1-11 of HA2 represent an important antigenic site on influenza virus.     

Monoclonal antibodies to the thyrotropin receptor raised by an autoantiidiotypic protocol and their relationship to monoclonal autoantibodies from Graves' patients

Monoclonal antibodies that bind to the TSH receptor were obtained by an autoantiidiotypic approach in which immunization of BALB/c mice was performed with mixtures of bovine (b) and human (h) TSH. Two of 28 positive wells were selected for cloning and characterization: D2 and 4G11. Their antiidiotypic character was evidenced by TSH-inhibitable binding to affinity-purified polyclonal anti-TSH. The specificity of D2 and 4G11 for the hormone-binding region of the TSH receptor was demonstrated by several findings: 1) they inhibited the binding of [125I]iodo-bTSH to receptor in a dose-dependent manner; 2) their binding to partially purified thyroid plasma membranes could be completely inhibited by bTSH and hTSH; and 3) they inhibited the TSH-dependent growth and adenylate cyclase stimulation in FRTL-5 cells in a dose-dependent manner. By Western blot analysis of bovine thyroid membranes, D2 bound to a polypeptide of 188,000-195,000 mol wt under nonreducing conditions and 54,000-59,000 mol wt after treatment of membranes with beta-mercaptoethanol; the 4G11 epitope was undetectable. Scatchard analysis of the binding of 125I-labeled antibodies to receptor showed that 4G11 bound to a single site with a Kd of 5.7 X 10(-9) M, whereas D2 showed complex binding characterized by high affinity (Kd = 1.74 X 10(-11) M) and low affinity (Kd = 1.3 X 10(-8) M) sites. Binding studies in which D2 and 4G11 competed with each other for the TSH receptor showed mutual but unequal inhibition. The data suggest that portions of the D2 and 4G11 epitopes overlap, but that there is a high affinity binding site(s) for D2 for which 4G11 competes less effectively. The binding of D2 and 4G11 to TSH receptor was inhibited by monoclonal antibodies secreted by Graves' heterohybridomas, showing that D2 and 4G11 share characteristics with autoantibodies of Graves' disease and lending support to the hypothesis that idiotypic network interactions may play a role in the pathogenesis of Graves' disease.     

Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor.

Two regions of human thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) (residues 12-44 and 308-364) were selected on the basis that they exhibit no sequence resemblance to luteinizing hormone/chorionic gonadotropin receptor. Five synthetic overlapping peptides (12-30, 24-44, 308-328, 324-344, and 339-364) were studied for their ability to bind 125I-labeled human TSH (hTSH), its isolated alpha and beta subunits, bovine TSH, ovine TSH, human luteinizing hormone, and human follicle-stimulating hormone. The human TSHR peptides 12-30 and 324-344 exhibited remarkable binding activity to human, bovine, and ovine TSH and to the beta chain of hTSH. Lower binding activity resided in the adjacent overlapping peptides, probably due to the contribution of the shared overlap to the binding. The specificity of TSH binding to these peptides was confirmed by their inability to bind human luteinizing hormone, human follicle-stimulating hormone, and the alpha chain of hTSH. Thyrotropins did not bind to bovine serum albumin or to peptide controls unrelated to the TSHR system. Furthermore, the binding of hTSH to TSHR peptides 12-30 and 324-344 was almost completely (approximately 90%) inhibited by rabbit antibodies against hTSH but not by antisera against unrelated proteins. It is concluded that the binding of TSH to its receptor involves extensive contacts and that the TSHR peptides 12-30 and 324-344 contain specific binding regions for TSH that might be either independent sites or two faces (subsites) within a large binding site.     

Clinical studies of an interferon inducer, polyriboinosinic-polyribocytidylic acid [poly (I)-poly (C)], in children.

Fifteen children ranging in age from one and one-half to 14 years received intravenous polyriboinosinic-polyribocytidylic acid [poly (I)-poly (C)], an interferon inducer. The patients all had serious neurologic illness either directly or circumstatially related to viral infection. Peak titers of interferon in serum ranged from 8 to 500 units/ml in response to doses of poly (I)-poly (C) of 0.1-1.0 mg/kg. Serum interferon persisted for less than or equal to 24 hr after induction. Hyporesponsiveness to a second dose of poly (I)-poly (C) persisted for seven days after initial induction, after which time full response again occurred. The half-life of poly (I) poly (C) in plasma as measured in three patients was less than 30 min. poly (I-poly(C) appears to be safe when given intravenously, but the low titers of interferon induced may limit its clinical usefulness as an antiviral drug.     

Poly(A)-poly(U) induces circulating colony-stimulating activity resulting from interactions between endogeneous interleukin 6 and serum components

The immunostimulant poly(A)-poly(U) induces a rapid enhancement of circulating colony-stimulating activity (CSA) in normal mice, culminating 2 h after i.v. injection. A dose of 200 micrograms per mouse is sufficient for a maximal effect. The colonies formed in response to sera from poly(A)-poly(U)-injected mice are mainly granulocytic with few macrophages. These sera are devoid of detectable interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF), but contain large amounts of interleukin 6 (IL-6) that are perfectly correlated with circulating CSA levels. Although, in our hands, IL-6 alone induces no colony formation in the standard methylcellulose colony assay, it is nevertheless requisite for this biological activity because 1) monoclonal antibodies against IL-6 strongly diminish colony formation in response to sera from poly(A)-poly(U)-injected mice, and 2) recombinant (r)IL-6 induces colonies when tested in combination with low amounts of normal murine serum. At the concentrations used (0.3%-2.5%), the latter has no or a very slight effect alone. Low amounts of hematopoietic growth factors, that is, macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), GM-CSF, or IL-3 that are almost ineffective in the absence of IL-6 can replace normal serum. Taken together, these data suggest that circulating IL-6, induced by i.v. injection of poly(A)-poly(U), promotes colony formation by interacting with serum components that might be identical with hematopoietic growth factors present in normal serum at subliminal concentrations. Finally, the involvement of lipopolysaccharide (LPS) in this phenomenon has been ruled out by the use of the low responder strain of mice (C3H/HeJ) that leads to similar results.