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Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H 2 O 2 after treatment but also after infection with B. cinerea . Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea . The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea , opening the door for the development of sustainable alternatives for chemical pesticides.

Single B cell antibody technology is a method for isolating antigen-specific B cells from human peripheral blood and obtaining antibody genes in developing antibody drugs. However, owing to immune tolerance to autoantigen, human autoantigen-specific B cells are difficult to acquire by conventional single B cell technology. In this study, we constructed a nitrated T-cell epitope named NitraTh by incorporating p -nitrophenylalanine into a universal T helper epitope. NitraTh had enhanced ability to activate CD4 + T cells and can be recognized by CD4 + T cells with different HLA class II haplotypes. This NitraTh can also break immune tolerance to autoantigens, such as human epidermal growth factor receptor 2 (HER2) and cannabinoid receptor 1, and induce strong specific IgM + B cell responses in vitro . HER2-NitraTh vaccine can also stimulate the generation of HER2-specific IgG + B cells in human immune system mice, which was established by cotransplanting lymphocytes and autologous dendritic cells in immunodeficient mice. We obtained 30 fully human IgG antibody genes by sorting single B cells from the human immune system mice immunized with HER2-NitraTh vaccine. The analysis of antibody genes showed that sorted B cells underwent the extensive somatic mutation of the antibody genes. We randomly selected eight genes for cloning, six of which expressed antibodies that can bind to HER2. Hence, we provided a convenient and effective method in acquiring fully human antibody genes against self-proteins, which can be used in developing therapeutic antibody drugs., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Liangliang Jiang et al.)

Objectives: Various rheumatoid arthritis (RA) HLA-DRB-1 risk haplotypes have been regrouped under the shared epitope (SE) in position 70-74. The presence of Valine in position 11 (Val11) and phenylalanine in position 13 (Phe13) are also associated with RA, but it is impossible to differentiate their role compared with the SE since they are in strong linkage disequilibrium (LD) in humans. Similar to humans, certain macaques express the SE (H6). We analysed the effect of various DRB1 haplotypes on T-cell response to citrullinated peptides (Cit-P) in macaques., Methods: Six H6 and six non-H6 macaques were immunized with four Cit-P. T-cell response was assessed using Interferon γ enzyme-linked immunospot., Results: Animals developed a specific anti-Cit-P T-cell response. Surprisingly, H6 animals had a significantly lower T-cell response than non-H6. In macaques, the 70-74 SE and the Val11 are on separate haplotypes. Presence of Val11 was strongly associated with the anti-Cit-P T-cell response, whatever the 70-74 sequence was. This response was amplified in case of presence of Phe13., Conclusion: The absence of LD between Val11 and SE in macaques allowed us to demonstrate that the most important HLA positions to induce a T-cell response against Cit-P were Val11 and Phe13 and not the 70-74 SE., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

In vitro immunization can to used to produce monoclonal antibodies(mAbs), but this technology is limited by poor reproducibility and the requirement of pre-immunized lymphocytes. To improve this approach, we recently developed a method for rapid generation of antigen-specific B cells. Here, we report the application of this system to the production of human IgGs against tumor necrosis factor (TNF). We expressed mutant proteins with site-specific incorporated p-nitrophenylalanine (pNO 2 Phe), which stimulated an in vitro immune response in human immune cells. After constructing an antigen-specific antibody library from in vitro immunized B cells identified by fluorescence-activated cell sorting, we demonstrated that many point mutation events of the variable region occurred in our step-by-step co-cultivation system for affinity maturation in vitro. To mimic the class switching, we panned for high-affinity antigen-binding fragments by the phage display method, assembled them and identified hTNF-neutralizing human IgGs. This approach may provide a general method for raising high-affinity monoclonal antibodies against self-proteins. Furthermore, it supports mechanistic understanding in breaking human self-tolerance with pNO 2 Phe.

Background: This study assessed the immunogenicity of pegvaliase (recombinant Anabaena variabilis phenylalanine [Phe] ammonia lyase [PAL] conjugated with polyethylene glycol [PEG]) treatment in adults with phenylketonuria (PKU) and its impact on safety and efficacy., Methods: Immunogenicity was assessed during induction, upward titration, and maintenance dosing regimens in adults with PKU (n = 261). Total antidrug antibodies (ADA), neutralizing antibodies, immunoglobulin (Ig) M and IgG antibodies against PAL and PEG, IgG and IgM circulating immune complex (CIC) levels, complement components 3 and 4 (C3/C4), plasma Phe, and safety were assessed at baseline and throughout the study. Pegvaliase-specific IgE levels were measured in patients after hypersensitivity adverse events (HAE)., Findings: All patients developed ADA against PAL, peaking by 6 months and then stabilizing. Most developed transient antibody responses against PEG, peaking by 3 months, then returning to baseline by 9 months. Binding of ADA to pegvaliase led to CIC formation and complement activation, which were highest during early treatment. Blood Phe decreased over time as CIC levels and complement activation declined and pegvaliase dosage increased. HAEs were most frequent during early treatment and declined over time. No patient with acute systemic hypersensitivity events tested positive for pegvaliase-specific IgE near the time of the event. Laboratory evidence was consistent with immune complex-mediated type III hypersensitivity. No evidence of pegvaliase-associated IC-mediated end organ damage was noted., Interpretation: Despite a universal ADA response post-pegvaliase administration, adult patients with PKU achieved substantial and sustained blood Phe reductions with a manageable safety profile. FUND: BioMarin Pharmaceutical Inc., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Recombinant antibody libraries can provide a source of renewable and high-performing binders tailored for use in affinity proteomics. In this context, the process of generating site-specific 1:1 tagging/functionalization and/or orientated surface immobilization of antibodies has, however, proved to be challenging. Hence, novel ways of generating such engineered antibodies for use in affinity proteomics could have a major impact on array performance. In this study, we have further tailored the design of human recombinant scFv antibodies for site-specific photocoupling through the use of an unnatural amino acid (UAA) and the Dock'n'Flash technology. In more detail, we have generated the 2nd generation of scFvs carrying the photoreactive UAA p-benzoyl-l-phenylalanine (pBpa). Based on key properties, such as expression levels, activity, and affinity, a preferred choice of site for pBpa, located in the beginning of the C-terminal affinity-tag, was for the first time pin-pointed. Further, the results showed that pBpa mutated antibody could be site-specifically photocoupled to free and surface immobilized β-cyclodextrin (an affinity ligand to pBpa). This paves the way for use of scFv antibodies, engineered for site-specific photochemical-based tagging, functionalization, and orientated surface immobilization, in affinity proteomics., (Copyright © 2017. Published by Elsevier B.V.)

Dry eye disease is an extremely common condition affecting millions worldwide. The underlying pathophysiological mechanism is thought to be localized inflammation of the ocular surface resulting in the localization of T cells at this surface followed by their activation and subsequent liberation of cytokines. This effect on T cells results from the binding of lymphocyte function-associated antigen-1 (LFA-1) located on T cells to intercellular adhesion molecule 1 (ICAM-1) expressed on inflamed epithelium and endothelium, and on T cells. Lifitegrast is a T-cell integrin antagonist designed to mimic ICAM-1, thus blocking the interaction of LFA-1 and ICAM-1. Lifitegrast enters the systemic circulation to a limited extent thus reducing the likelihood of unwanted systemic reactions. Clinical trials in over 2,500 subjects with dry eye disease have shown that 5.0% lifitegrast given by ocular instillation causes a significant reduction in objective and subjective signs and symptoms of the disease. These beneficial effects are associated with a relatively low incidence of unwanted effects, almost all local in nature. In light of these findings, lifitegrast was approved by the Food and Drug Administration (FDA) in 2016 for the treatment of dry eye disease, the first drug with this mechanism of action to be so approved., (Copyright 2016 Prous Science, S.A.U. or its licensors. All rights reserved.)

In this study, we prepared polyclonal antibodies against anti-diabetic drug nateglinide (NTG), and established a sensitive chemiluminescent immunoassay (CLIA) to detect NTG in tablets and serum. Two kinds of immunogens were synthesized using ethylcarbodiimide (EDC)/hydroxysuccinimide (NHS) and carbonyldiimidazole (CDI)/4-dimethylaminopyridine (DMAP) as coupling reagents respectively. When activated by EDC/NHS, more molecules of NTG coupled with carrier protein in immunogens. A horseradish peroxidase (HRP)-luminol-H2O2 system with p-iodophenol enhancement was applied in the CLIA analysis. The antibodies in EDC/NHS group showed higher titer, sensitivity and wider detection linear range than those in CDI/DMAP group, and were chosen for next studies. The developed CLIA assay exhibited good selectivity towards NTG among structually similar analogs. The method could detect as low as 0.35 ng mL(-1) NTG in buffer, 2.1 ng mL(-1) NTG in serum and 0.84 ng mL(-1) NTG in tablets. The CLIA method provided consistent results with HPLC method (r=0.9986) in determination of NTG from 5.0 to 400 µg mL(-1). The CLIA method could detect 78 samples in one assay, and the samples need only dilution in pretreatment. As a summary, this research offers a sensitive assay for high-throughout screening of NTG in formulation control and pharmacokinetic studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

PIWIL2, a member of PIWI/AGO family, is expressed in germline stem cells and precancerous stem cells, but not in adult somatic cells. PIWIL2 plays an important role in tumor development. It is considered as a cancer–testis antigen (CT80). It has been reported that the spliced fragment of PIWIL2, PL2L60, was widely expressed in cancer cell lines. In this study, HLA-A2-restricted epitopes from PL2L60 were predicted by online tools. To improve the activity of the native epitope, a candidate peptide P281 with potent binding affinity was chosen to investigate the modification strategy. A series of aromatic amino acids were introduced to substitute the first residue of P281. Then, we tested the binding affinity and stability of the peptide analogs and their ability to elicit specific immune responses both in vitro and in vivo. Our results indicated that the cytotoxic T lymphocytes (CTLs) induced by [4-Cl-Phe1]P281 could elicit more potent activities than that of P281 and other analogs. The CTLs induced by this analog could lyze target cells in HLA-A2-restricted and antigen-specific manners. [4-Cl-Phe1]P281 also showed the best resistance against degradation in human serum. In conclusion, the introduction of the unnatural amino acid, 4-Cl-Phe, into the first position could enhance the activity of the native epitope to induce cytotoxic T lymphocytes. It might be a good strategy to modify other promising native epitopes. The novel epitopes identified in this study could be used as novel candidates to the immunotherapy of HLA-A2 positive patients with tumors expressing PL2L60.

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 μM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

L-amino acid oxidases (LAAO) are flavoproteins that catalyze the oxidative deamination of L-amino acids to a keto-acid along with the production of H₂O₂ and ammonia. Interleukin 4 induced gene 1 (IL4I1) is a secreted LAAO expressed by macrophages and dendritic cells stimulated by microbial derived products or interferons, which is endowed with immunoregulatory properties. It is the first LAAO described in mammalian innate immune cells. In this work, we show that this enzyme blocks the in vitro and in vivo growth of Gram negative and Gram positive bacteria. This antibiotic effect is primarily mediated by H₂O₂ production but is amplified by basification of the medium due to the accumulation of ammonia. The depletion of phenylalanine (the primary amino acid catabolized by IL4I1) may also participate in the in vivo inhibition of staphylococci growth. Thus, IL4I1 plays a distinct role compared to other antibacterial enzymes produced by mononuclear phagocytes.

Stimulation via the T-cell receptor (TCR) activates p38α and p38β by phosphorylation of p38 Tyr-323 (p38(Y323)). Here we characterize knockin mice in which p38α and/or β Tyr-323 has been replaced with Phe. We find that p38α accounts for two-thirds and p38β the remainder of TCR-induced p38 activation. T cells from double knockin mice (p38αβ(Y323F)) had defects in TCR-mediated proliferation and Th1 and Th17 skewing, the former corresponding with an inability to sustain T-bet expression. Introduction of p38α(Y323F) into Gadd45α-deficient mice, in which the alternative p38 pathway is constitutively active, reversed T-cell hyperproliferation and autoimmunity. Furthermore, p38αβ(Y323F) mice had delayed onset and reduced severity of the inflammatory autoimmune diseases collagen-induced arthritis and experimental autoimmune encephalomyelitis. Thus, T cell-specific alternative activation of p38 is an important pathway in T-cell proliferation, Th skewing, and inflammatory autoimmunity, and may be an attractive tissue-specific target for intervention in these processes.

The site-specific incorporation of the unnatural amino acid p-nitrophenylalanine (pNO(2)Phe) into autologous proteins overcomes self-tolerance and induces a long-lasting polyclonal IgG antibody response. To determine the molecular mechanism by which such simple modifications to amino acids are able to induce autoantibodies, we incorporated pNO(2)Phe, sulfotyrosine (SO(3)Tyr), and 3-nitrotyrosine (3NO(2)Tyr) at specific sites in murine TNF-α and EGF. A subset of TNF-α and EGF mutants with these nitrated or sulfated residues is highly immunogenic and induces antibodies against the unaltered native protein. Analysis of the immune response to the TNF-α mutants in different strains of mice that are congenic for the H-2 locus indicates that CD4 T-cell recognition is necessary for autoantibody production. IFN-γ ELISPOT analysis of CD4 T cells isolated from vaccinated mice demonstrates that peptides with mutated residues, but not the wild-type residues, are recognized. Immunization of these peptides revealed that a CD4 repertoire exists for the mutated peptides but is lacking for the wild-type peptides and that the mutated residues are processed, loaded, and presented on the I-A(b) molecule. Overall, our results illustrate that, although autoantibodies are generated against the endogenous protein, CD4 cells are activated through a neo-epitope recognition mechanism. Therefore, tolerance is maintained at a CD4 level but is broken at the level of antibody production. Finally, these results suggest that naturally occurring posttranslational modifications such as nitration may play a role in antibody-mediated autoimmune disorders.

Glycolate oxidase (GO) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) are the two enzymes that serve key functions in the photorespiration and photosynthesis of plants. A 2 kDa highly basic phenylalanine-rich oligo-peptide (BOP) binds to the surface of acidic GO via ionic and hydrophobic interactions, forming the GO-BOP complex (GC). Previously, RubisCO was thought to exist as a single species composed of a large (rbc L, 54 kDa) and a small subunit (rbc S, 14 kDa). Here we show for the first time, using 2-DE, SDS-PAGE, immunoassays and amino acid determination, that BOP also interacts with RubisCO and that many RubisCO-BOP complexes (RCs), differing in pI, hydrophobicity and activity, coexist in green leaves. GCs, RCs and crude extract from green leaves analyzed by SDS-PAGE Western blotting showed that BOP exists either in subunit-BOP complexes (GO subunit-BOP, rbc L-BOP and rbc S-BOP etc.), with a wide variation in the number and the position of BOPs bound to each subunit molecular, or alone without a binding partner. When rbc L-BOP and rbc S-BOP were assayed by SDS-PAGE, BOP was dissociated from the subunit and it self-assembled to form 37 different BOP polymers (basic phenylalanine-rich protein) whose molecular weights (M(r)s) ranged from 34.0 to 91.6 kDa, indicating that the M(r) of BOP is about 2 kDa. Thus, the addition of BOP changes the M(r) of the subunit-BOP complexes so minimally that the rbc L and rbc S run at their predicted M(r)s on SDS-PAGE. In summary, the results described here demonstrate that the presence of BOP in complexes (both subunit-BOP complex and protein-BOP complex) can cause cross-reactivity of antibodies against different proteins., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The binding site of a monoclonal anti-L-amino acid antibody (anti-L-AA) was modeled using the program SWISS-MODEL. Docking experiments with the enantiomers of phenylalanine revealed that the antibody interacts with L-phenylalanine via hydrogen bonds and hydrophobic contacts, whereas the D-enantiomer is rejected due to steric hindrance. Comparison of the sequences of this antibody and an anti-D-amino acid antibody (anti-D-AA) indicates that both immunoglobulins derived from the same germline progenitor. Substitution of four amino acids residues, three in the framework and one in the complementarity determining regions (CDRs), allowed in silico conversion of the anti-L-AA into an antibody that stereoselectively binds D-phenylalanine.

A vacuolar serine protease (Pen ch 18) has been identified as a major allergen of Penicillium chrysogenum. The molecular features of antigenic determinant(s) on Pen ch 18 recognized by human IgE antibodies, however, have remained unclear. Here, we show that a dominant IgE epitope on the N-terminally processed Pen ch 18 allergen was narrowed down to residues 83-91. In addition, Lys89, Lys90, and possibly Phe91 were identified as the core residues. Substitution of Lys89, Lys90, or Phe91 with alanine can significantly reduce IgE-binding to Pen ch 18. Immunoblot inhibition confirmed that Lys89 and Phe91 played a significant role in IgE-binding against Pen ch 18. Molecular modeling suggests they are located on a loop-like structure at or near the surface of the major fungal allergen.

Background: Raising selectivity to tumor cells is a major challenge for most chemotherapy drugs. One of approaches to realizing this goal is antibody-directed enzyme prodrug therapy (ADEPT). This study was done to investigate the curative effect of a new ADEPT system for the treatment of prostate cancer., Methods: Methotrexate (MTX) prodrugs were synthesized and anti-seminoprotein (SM) single-chain antibody/human carboxypeptidase-A fusion protein (scFv/hCPA) was prepared. Therapeutic effects of this ADEPT system were evaluated., Results: The synthesis of prodrugs was successful and the prodrugs were confirmed no cytotoxicity, but hydrolysis with tumor-targeted scFv/hCPA fusion protein gave 1,000-fold higher cytotoxicity than MTX-alpha-Phe only. Cell cycle assays showed that tumor cells were arrested in the S phase after ADEPT treatment; furthermore, tumors were inhibited significantly in scFv/hCPA and MTX-alpha-Phe treated mice., Conclusions: Our results suggest that targeted activation cytotoxicity against established prostate cancer by scFv/hCPA mediated ADEPT is tumor-specific and has no systemic toxicity in vitro and in vivo.

1. We investigated the effect of bisphenol A (BPA), which binds estrogen receptors, on immune responses including production of antigen-specific antibodies, proliferative responses of lymphoid cells, and Th1 and Th2 responses. 2. For this investigation, mice were p.o. given varying doses including 3, 30, 300, and 3000 micro g kg(-1) of BPA immediately after immunization with hen egg lysozyme (HEL) (day 0) and then daily by day 20. On day 21, anti-HEL IgG antibodies in sera and proliferative responses of spleen cells to the antigen were measured. Anti-HEL IgG2a antibodies and IFN-gamma secreted from splenic lymphocytes were also measured as indicators of Th1 immune responses, while anti-HEL IgG1 antibodies and IL-4, as those of Th2 responses. 3. The results showed that treatment with 3000 micro g kg(-1) of BPA was followed by a significant increase in anti-HEL IgG as well as the antigen-specific cell proliferation. Anti-HEL IgG2a production and IFN-gamma secretion were significantly enhanced in mice treated with 300 and 30 micro g kg(-1) of BPA, respectively, while anti-HEL IgG1 production and IL-4 secretion were augmented in animals given 3000 and 300 micro g kg(-1) of the chemical, respectively. 4. Augmentation of these immune responses was also observed in mice exposed to 0.3-30 micro g kg(-1) of estradiol, although Th1 responses appeared to be more sensitive to the sex hormone than Th2 responses. 5. These results suggest that BPA may play a role in augmenting immune responses, especially Th1 responses.

We determined if a single amino acid substitution in a self protein causes autoantibody responses. Mouse lysozyme (ML) was used as a model self protein, and a mutant ML (F57L ML) was prepared by replacing 57Phe of ML to Leu, an approach which resulted in introducing into ML the immunogenic sequence of peptide 50-61 of hen egg lysozyme (HEL) restricted to I-A(k) MHC class II molecule. We found that F57L ML but not native ML primed HEL specific T cells and triggered ML specific autoantibody responses in B10.A and C3H mice (I-A(k), I-E(k)). Peptide regions, ML 14-69 and ML 98-130, were major epitopes of autoantibodies in both strains of mice. These findings indicate that a single amino acid substitution in self proteins can cause an autoantibody response when the mutated region is presented by MHC class II molecules and recognized by T cells.

The present study was an attempt to map the epitope in P1 protein which may cross-react with anti-dsDNA. In addition to wild-type P1, the genes of its C-terminal mutants were cloned and expressed. The binding activity of these proteins with anti-dsDNA was determined by Western blot and ELISA. The P1 mutants with complete deletion of the acidic charge and hydrophobic clusters, deletion of the hydrophobic cluster, or replacement of the phenylanlanines with alanine in the hydrophobic cluster lost the binding activity. Moreover, P1 mutants with mutation at the serine phosphorylation sites (positions 102 and 105) retained their binding activities with anti-dsDNA. However, those with mutation at the serine phosphorylation sites and without the hydrophobic cluster lost their binding activities. These findings suggest that phenylalanines in the C-terminal hydrophobic cluster region of ribosomal P proteins are essential to their cross-reactivity with anti-dsDNA., (Copyright 1999 Academic Press.)

Antibody IgE Lb4 interacts favorably with a large number of different compounds. To improve the current understanding of the structural basis of this vast cross-reactivity, the binding of three dinitrophenyl (DNP) amino acids (DNP-alanine, DNP-glycine, and DNP-serine) is investigated in detail by means of docking and molecular dynamics free energy simulations. Experimental binding energies obtained by isothermal titration microcalorimetry are used to judge the results of the computational studies. For all three ligands, the docking procedure proposes two plausible subsites within the binding region formed by the antibody CDR loops. By subsequent molecular dynamics simulations and calculations of relative free energies of binding, one of these subsites, a tyrosine-surrounded pocket, is revealed as the preferred point of complexation. For this subsite, results consistent with experimental observations are obtained; DNP-glycine is found to bind better than DNP-serine, and this, in turn, is found to bind better than DNP-alanine. The suggested binding mode makes it possible to explain both the moderate binding affinity and the differences in binding energy among the three ligands.

The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.

Myelin basic protein (MBP)-reactive T cells may play an important role in the pathogenesis of multiple sclerosis (MS). The T cell response to the 83-99 region of MBP represents a dominant autoreactive response to MBP in MS patients of DR2 haplotype. In this study, a large panel of DR2- and DR4-restricted T cell clones specific for the MBP83-99 peptide were examined for the recognition motifs and structural requirements for antigen recognition using alanine-substituted peptides. Our study revealed that although the recognition motifs of the T cell clones were diverse, the TCR contact residues within the 83-99 region of MBP were highly conserved. Two central residues (Phe90 and Lys91) served as the critical TCR contact points for both DR2- and DR4-restricted T cell clones. Single alanine substitution at residue 90 or residue 91 abolished the responses of 81-95 % of the T cell clones while a double alanine substitution rendered all T cell clones unresponsive. It was also demonstrated in this study that the substituted peptides altered the cytokine profile of some, but not all, T cell clones. Some MBP83-99-specific T cell clones were able to sustain alanine substitutions and were susceptible to activation by microbial antigens. The study has an important implication in designing a peptide-based therapy for MS.

Shortage of reliable plasma assays has hampered studies of cholecystokinin (CCK). The assay problems are low plasma concentrations, extensive molecular heterogeneity, and close homology of CCK to gastrin, which circulates in higher concentrations. To develop an accurate CCK RIA, antibodies were raised in rabbits, guinea pigs, and mice in titers from 200 to 4000000. The specificity of the antisera was tested with homologous peptides, and tissue and plasma extracts. Rabbit 92128 produced antibodies in high titer (> or =500000) with sufficient avidity (K(o)eff > or = 10(12) mol(-1)) and the desired specificity. The antiserum binds the bioactive forms of CCK with equimolar potency and displays no reactivity with gastrin. CCK concentrations in plasma from healthy humans rose from 1.13 +/- 0.10 pmol/L (mean +/- SE, n = 26) to 4.92 +/- 0.34 pmol/L after a mixed meal. Chromatography of human plasma revealed traces of CCK-58, a predominance of CCK-33 and CCK-22, and moderate amounts of CCK-8. The results show that it is possible to produce specific CCK-antisera using a sulfated CCK-12 analog.

HLA-B*3501 and -B*5101 molecules, which belong to the HLA-B5 cross-reactive group, bind peptides carrying similar anchor residues at P2 and the C-terminus, but differences are observed in the preference for a Tyr residue at the C-terminus and the affinity of peptides. A recent study of HLA-B*3501 crystal structure suggested that residue 116 on the floor of the F-pocket determines a preference for anchor residues at the C-terminus. In order to evaluate the role of the residue 116 in the peptide binding to both HLA-B*3501 and HLA-B*5101 molecules, we generated HLA-B*3501 mutant molecules carrying Tyr at residue 116 (B*3501-116Y) and tested the binding of a panel of nonamer peptides to the B*3501-116Y molecules by a stabilization assay with RMA-S transfectants expressing the mutant molecules. The substitution of Tyr for Ser at residue 116 markedly reduced the affinity of nonamer peptides carrying Tyr at P9, while it enhanced that of nonamer peptides carrying Ile and Leu at P9. On the other hand, the affinity of peptides carrying aliphatic hydrophobic residues at P9 to B*3501-116Y molecules was much higher than that to HLA-B*3501 and HLA-B*5101 molecules. These results indicate that residue 116 is critical for the structural difference of the F-pocket between HLA-B*3501 and HLA-B*5101 which determines the C-terminal anchor residues, while leaving other residues which differ between HLA-B*3501 and HLA-B*5101 may be responsible for the low peptide binding property of the latter.

Two closely-related molecules, DR(alpha,beta1*0101) and DR(alpha,beta1*0103), whose beta chains only differ by three amino acids at positions 67, 70, and 71, and six intermediate molecules obtained by site-directed mutagenesis were used to ascertain the respective roles of the three polymorphic residues. Substitutions at positions 70 (D-->Q), 71 (E-->R) and 67 (I or L-->F) strongly affected HA 306-318-specific T-cell recognition. The consequences of the substitution of residue 67 by a phenylalanine depended on the modified HLA-DR molecule. Although this substitution completely inhibited peptide-specific DR1-restricted T-cell recognition, its manifestations on the DR103-restricted T-cell response were variable (abolishing proliferation of some cell lines and not others), no matter what the peptide presented was (HA 306-319 or HIV P25 peptides). We also observed that inhibition of the proliferation of an alloreactive anti-DR103 T-cell clone, caused by a substitution at position 70, was completely cancelled by substitution of residue 67 by a phenylalanine. The observations based on functional experiments, thus, suggest that residue 67 plays an important role in determining conformation of the peptide presented to the T cells. Molecular modeling was used to predict changes induced by amino acid substitutions and highly supports functional data. Substitution of residue 67 by a phenylalanine could have repercussions on the structure of HLA-DR molecule/peptide complexes and affect T-cell recognition.

Immobilization of low molecular weight haptens in ELISA usually involves their coupling to protein molecules or covalent binding to the solid phase. In this study we demonstrate that it is possible to directly bind the hapten p-aminophenylalanine to gamma-irradiated polystyrene microtiter plates for the detection of antibodies that stereospecifically recognize the chiral center of alpha-amino acids. Simple incubation of the hapten in aqueous buffer, without additional activation, results in a stable coating that is suited for use in ELISA.

The adsorbability of pemphigus vulgaris (PV) antibodies to immunoadsorbent with phenylalanine, tryptophan, and dextran sulfate as ligands was examined. PV antibodies were adsorbed to immunoadsorbents with both phenylalanine and tryptophan as ligands, in batchwise adsorption and column adsorption. PV antibodies were not adsorbed to immunoadsorbent with dextran sulfate as ligand, in column adsorption. It was also shown that the rates of decrease of normal protein fractions in column adsorption with phenylalanine as a ligand were small. Our results suggest that immunoadsorption therapy using sorbent with phenylalanine as a ligand may be useful for the treatment of PV.

The random amino acid copolymer (Glu60, Phe40)n (GPhe) was previously shown to augment antigen-dependent proliferation of the murine TH2 cell lines DCL-2 and D10.G4.1. In the present study, the addition of GPhe to (Glu36, Lys24, Ala40)n (GLA)-primed BALB/c primary lymph node (1 degree LN) T cell cultures, the source of DCL-2, resulted in significant suppression of both the proliferative and lymphokine response to GLA. Suppression by GPhe of the 1 degree LN response was subsequently shown to be neither antigen- nor haplotype restricted, and was inhibitable by polyclonal anti-GPhe antibodies. Studies were extended to a GLA-reactive T cell hybridoma clone (DL.4G6.1). where significant suppression by GPhe of GLA-stimulated lymphokine production was observed as measured by markedly decreased HT-2 stimulatory activity of the collected supernatants. Subsequent antibody blocking experiments employing the monoclonal anti-murine IL-4 antibody 11B11 revealed that BALB/c GLA-reactive 1 degree LN T cells and DL.4G6.1 did not produce detectable levels of IL-4 in their culture fluids when stimulated by GLA, which suggested that these cells, unlike DCL-2, were TH1-like in nature. The addition of GPhe to the TH1 clones 5.2 and 5.9 resulted in significant suppression of proliferation to homologous antigen (ovalbumin), in contrast to the augmentation observed with the TH2 cell lines DCL-2 and D10.G4.1. It was concluded from these data, that the addition of GPhe to various T cell cultures lead to unusual suppressive and augmenting activities specific for TH1 and TH2 cells, respectively. Although the mechanism for these dichotomous effects of GPhe is as yet undetermined, several possibilities are considered.

Following interaction of the random polymer (Glu60,Phe40)n (GPhe) with antigen-presenting accessory cells (APC), unusual costimulatory activities were noted in several murine T cell systems. When GPhe, in contrast with other random copolymers (GT,GL), was added during "inhibition" and T cell "repertoire" studies as a (negative) control to GLA-reactive nonclonal T cell lines of haplotypes H-2d (DCL-2) or H-2bm12, augmentation of T cell proliferation ([3H]thymidine incorporation ([3HT]) to homologous antigen was observed. Augmentation by GPhe was also observed in the response of a GLPhe-reactive (H-2s X H-2d)F1 T cell line and the allogeneic response of the clonal T cell line D10.G4.1. This augmentation was critically dependent on the concentration of adherent accessory cells. Although the mechanism of action of GPhe remains, as yet, undefined, the GPhe-mediated enhancement of DCL-2 (a TH2, H-2d anti-GLA, T cell line) proliferation was not dependent upon the production of either IL-1 or IL-6 by accessory cells. In addition, enhanced DCL-2 proliferation was not accompanied by a significant increase in detectable IL-4 release.

Our previous study (1) demonstrated the "cytokine-like" activity of poly(Glu60,Phe40)(GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu36,Lys24,Ala40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 +/- IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (+/- IL-2, +/- IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (+/- IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface "adhesion" molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF alpha, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.

The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy. The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA). Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin. The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide. Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA). These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody. The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide. Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide. In the bound peptide, we observe interactions of a lysine residue with aspartate-10 beta protons. While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to investigate the compound 4-nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline (NOPYE) and some related compounds for skin sensitization in guinea pigs, as the first step in a search for more effective skin sensitizers for immunotherapy of cutaneous tumors. In guinea pigs, NOPYE and NOPYE-L-alanine produce far milder delayed hypersensitivity reactions than DNCB. Both NOPYE and DNCB fail to act as adjuvants for skin sensitization to tuberculin purified protein derivative (PPD) and ovalbumin (OV). This suggests an explanation for the lack of effectiveness of DNCB in immunotherapy of metastases: DNCB may be relatively ineffective as an adjuvant for production of specific antitumor immunity. Such adjuvant activity may be essential if the action of the immunotherapeutic reagent is not to be confined to its site of application but is to be effective at the site of distant metastases.

Several wild-derived H-2 haplotype mice were recently shown by serology and tryptic peptide fingerprinting to express I-region A molecules closely related to the Ap and Ak molecules of the laboratory strains. To determine if such naturally occurring minor structural variations in the A molecule alter Ir gene-controlled responsiveness, we examined the immune responses of these strains after primary immunizations to three synthetic polypeptide antigens: G60Phe40, GLPhe9, and GAT10. Inbred strains carrying the Ap (or Aq) allele are known to be high responders to G60Phe40 and GLPhe9 but low responders to GAT10. Strains expressing the Ak allele are classified as low responders to G60Phe40 and GLPhe9, but high responders to GAT10. Of seven strains examined belonging to the Ap family, one (B10.CAS2) failed to respond to either G60Phe40 or GLPhe9 as measured by antibody production and T cell proliferation. In addition, two strains (B10.STC90 and W12A) of the Ak family were found to be of responder phenotype to GLPhe9. Both GLPhe9 responses resulted from the introduction of new E beta genes into the I region through naturally occurring intergenic recombination between A beta A alpha and E beta. All strains of mice in the Ak family proved to be of the high responder phenotype in their responses toward GAT10. These results contrast strongly with known patterns of alloreactivity against the variant Ap and Ak molecules.

We developed an idiotypic radioimmunoassay system that detects shared idiotypic determinants, termed GTGL idiotype, on antibodies bearing distinct antigen-binding specificities in various mouse strains. Either poly-(Glu, Tyr) (GT)- or poly-(Glu, Lys) (GL)-related determinants are able to induce anti-GT and anti-GL (GTGL)-idiotypic antibodies. Strain distribution studies indicate that GTGL-idiotypic antibodies are readily induced and frequently expressed in antisera obtained from 25 different mouse strains immunized either with GT-related or GL-related polymers. The ability to express GTGL-idiotypic antibodies is a dominant trait and is controlled by Igh-linked gene(s). In addition, we demonstrated that in anticopolymer of L-glutamine acid60- L-alanine30-L-tyrosine (GAT) and anticopolymer of L-glutamic acid54-L-lysine35-L-phenylalanine11(GLphi) antisera, both antibodies uniquely specific to GAT or GLphi, respectively, and antibodies bearing dual specificities for GAT and GLphi, expressed GTGL idiotype. The genetic implications of these findings are discussed. X

The possibility that the two complementing alpha- and beta-Ir-GLphi genes are independently responsible for controlling events in T lymphocytes and B lymphocytes, respectively, has been tested in double adoptive transfer experiments utilizing cells from appropriate inbred strains of mice. The results of these studies show that the functions of T lymphocytes and B lymphocytes and the cooperative interactions between T and B cells require the presence of both alpha- and beta-genes in each respective cell type. Moreover, evidence has been obtained in these studies that indicates a preference for the alpha- and beta-Ir-GLphi genes in the cis position to obtain the most effective T-B-cell interactions. The possible implications of these findings are discussed.

The antigenic heterogeneity of angiotensin II (AII) was studied with monoclonal antibodies. Twelve antibodies were produced and characterized. Association constants for AII varied from 1.2 X 10(8) to 1.1 X 10(10) M-1. The fine specificity of the Mab was studied by immunoenzymoassay using solid-phase AII. Using AII analogues in binding inhibition experiments, three groups of specificity could be characterized: (1) five antibodies reacted only with peptides in which phenylalanine is the carboxy terminal aminoacid; for two of these antibodies, tyrosine4 is closely associated with the binding site, since iodine labelling suppresses reactivity; (2) two antibodies also required phenylalanine in position 8, but, in addition, reacted with AI, a decapeptide in which phenylalanine is not terminal; (3) five antibodies reacted with analogues in which phenylalanine had been substituted for another amino acid. In addition, studies in which binding of a biotinylated Mab to solid-phase AII was analysed in the presence of various unlabelled Mab suggest further antigenic heterogeneity of AII.

The immune response (Ir) to the random copolymer GLphi depends upon the function of two Ir genes, Ir-GLphi-beta[beta] and Ir-GLphi-alpha[alpha], mapped to the I-A and I-E/C subregions of the major histocompatibility complex, respectively. In this paper, the site(s) of expression of the products of these two Ir genes was examined by evaluating T-lymphocyte proliferative responses of bone marrow radiation chimeras. Chimeras were created in [alpha+beta- X alpha-beta+]F1 responder mice by lethal irradiation and reconstitution with a mixture of bone marrow cells from both parental strains. These chimeras failed to respond to GLphi, although they were capable or responding to the much weaker antigens, (T,G)-A--L, TEPC-15, pigeon cytochrome c, and (H,G)-A--L. This failure to respond to GLphi was shown not to be the result of a cryptic mixed lymphocyte reaction, as similar chimeras created in (alpha+beta+ X alpha-beta+)F1 mice responded well to GLphi, although they possessed almost the same potential histoincompatibility. Furthermore, the lack of response to GLphi could not be attributed to a general failure of the two parental cell types in the chimeras to collaboratc with each other, as each chimeric parental cell type could respond to dinitrophenyl conjugated ovalbumin presented on nonimmune spleen cells from the other parent. Thus, the failure of low responder parental into F1 high responder chimeras to generate an immune response to GLphi suggests that immune competence for this antigen requires at least one cell type in the immune system to express gene products of both the Ir-glphi-alpha and -beta genes, i.e. one cell must be of high responder genotype. The the antigen-presenting cell is one such cell type was shown by experiments in which GLphi-primed T lymphocytes from responder F1 mice were stimulated with antigen bound to nonimmune spleen cells. Only spleen cells from responder F1 and recombinant mice could present GLphi. Neither of the two complementing nonresponder parental spleen cell populations, either alone or mixed together, could present GLphi, although both could present purified protein derivative of tuberculin. This was shown to be the case for T cells positively selected in vitro as well as freshly explanted T cells. Thus, both Ir-GLphi-alpha and Ir-GLphi-beta gene products must be expressed in the same antigen-presenting cell to generate a T-lymphocyte proliferative response to GLphi. The implications of these findings for models of two gene complementation are discussed.

An analysis of the effect of eleven monoclonal antibodies on the functional characteristics of monkey liver phenylalanine hydroxylase is presented. These eleven antibodies have been found to react with eight distinct regions on the phenylalanine hydroxylase protein. PH1 antibody inhibits enzyme activity, is dependent on phenylalanine for its binding, and appears to be related to structural changes occurring during phenylalanine activation of the enzyme activity. PH2 and PH3 antibodies stimulate enzyme activity, their binding is inhibited by lysolecithin and this group apparently is recognizing structures involved in lysolecithin activation of the enzyme activity. PH5, PH10, PH12 and PH6 recognise sites on phenylalanine hydroxylase affected by lysolecithin activation.

The synthetic random copolymer poly( Glu60 , Phe40 ) ( GPhe ) is an excellent immunogen in SWR/J (H-2q) mice. The intravenous injection of soluble GPhe by itself led to antibody production and GPhe -specific T-lymphocyte proliferation. The proliferating lymphocyte was sensitive to anti-Thy 1.2 and anti-Ly 1 antisera. A subsequent immunization with GPhe should therefore lead to an enhanced response. Yet, a single intravenous injection of an aqueous solution of GPhe suppressed the immune response to a subsequent immunization with GPhe in CFA as measured by GPhe -specific plaque-forming colonies, ELISA, T-cell proliferation, and delayed-type hyper-sensitivity. The suppression was not transferable from pretreated mice into normal or irradiated syngeneic recipients with either sera or lymphoid cells. The antibody generated from the pretreatment could not be responsible for the suppression as injection of SWR anti- GPhe antibody into SWR/J mice enhanced, rather than suppressed, the response to the subsequent immunization with GPhe . Pretreatment of mice with a rabbit anti-idiotypic antiserum produced against (SWR anti- GPhe antisera) had no effect on the immune response to GPhe . Thus, the suppression cannot be explained by a simple B-cell tolerance mechanism. This type of unusual suppression was observed only with mice of H-2q haplotype and not with mice of H-2 haplotypes a and k which are also responders to GPhe .

The antibody response to poly-(Glu53Lys36Phe11) (GLphi) has been shown to be under the control of two independent, major histocompatibility-linked immune response genes, designated alpha and beta. In the present work we demonstrate that the T-lymphocyte proliferative response is also under the control of these two immune response genes. Thus, mice of the H-2a, H-2b, H-2k, and H-2s haplotypes were all nonresponders to GLphi. In contrast F1 hybrids between these strains, such as (B10 X B10.A)F1 and (C3H X SJL)F1, as well as several recombinant mice derived from the nonresponder haplotypes, such as B10.1(5R), B10.HTT, and B10.S(9R), were all responders to GLphi. The complementation between nonresponder genomes appeared to be stronger in the cis position than in the trans position for some strain combinations. The failure of strains bearing only one of the two responder alleles to show a T-lymphocyte proliferative response to GLphi, argues strongly that neither gene can be expressed exclusively in B lymphocytes. This conclusion is discussed in relation to another two gene model which has recently been proposed.