AB Interaction Research Articles

Alanine scanning mutants of rat proinsulin I show functional diversity of anti-insulin monoclonal antibodies.

In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.

High affinity of anti-GBM antibodies from Goodpasture and transplanted Alport patients to α3(IV)NC1 collagen

Anti-glomerular basement membrane (anti-GBM) antibody-mediated diseases are characterized by rapidly progressive glomerulonephritis (RPGN) that often results in irreversible loss of renal function and renal failure. Although many factors contribute to the fulminant nature and treatment resistance of this disease, we questioned whether high affinity autoantibody-alpha3(IV) collagen interactions lead to persistent antibody deposition, thereby perpetuating inflammation. To address this hypothesis, the binding kinetics of human anti-GBM antibodies (Ab) to alpha3(IV)NC1 were evaluated using an optical biosensor interaction analysis. Polyclonal anti-GBM Abs were purified by alpha3(IV)NC1 affinity chromatography from the sera of patients with anti-GBM AB-mediated diseases, including individuals with Goodpasture syndrome (GS), idiopathic RPGN (N = 7), and Alport syndrome (AL) following kidney transplantation (N = 4). The affinity-binding characteristics of the autoantibodies were determined using a biosensor analysis system, with immobilized bovine alpha3(IV)NC1 dimers. All of the autoantibody preparations bound to alpha3(IV)NC1, whereas none bound to alpha1(IV)NC1 (control). Purified, normal serum IgG did not bind to either antigen. Estimated dissociation constants (Kd) for the purified autoantibodies were 1.39E-04 +/- 7.30E-05 s-l (GS) and 8. 90E-05 +/- 2.80E-05 s-l (AL). Their estimated association constants (Ka) were 2.67E+04 +/- 1.8E+04 (M-ls-l) and 2.76E+04 +/- 1. 70E+04(M-ls-l) for GS and AL patients, respectively. By comparison with other Ab interactions, these Abs demonstrated high affinity, with relatively high on (binding) rates and slow off (dissociation) rates. The results suggest that anti-GBM Abs bind rapidly and remain tightly bound to the GBM in vivo. This property likely contributes to both the fulminant nature of this disease and its resistance to therapy, because persistent glomerular Ab deposition has the potential to produce continuous inflammation, despite removal of circulating Abs and adequate immunosuppression.

Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1.

The symptoms characteristic of allergic hypersensitivity are caused by the release of mediators, i.e., histamine, from effector cells such as basophils and mast cells. Allergens with more than one B cell epitope cross-link IgE Abs bound to high affinity FcepsilonRI receptors on mast cell surfaces leading to aggregation and subsequent mediator release. Thus, allergen-Ab complexes play a crucial role in the cascade leading to the allergic response. We here report the structure of a 1:1 complex between the major birch pollen allergen Bet v 1 and the Fab fragment from a murine monoclonal IgG1 Ab, BV16, that has been solved to 2.9 A resolution by x-ray diffraction. The mAb is shown to inhibit the binding of allergic patients' IgE to Bet v 1, and the allergen-IgG complex may therefore serve as a model for the study of allergen-IgE interactions relevant in allergy. The size of the BV16 epitope is 931 A2 as defined by the Bet v 1 Ab interaction surface. Molecular interactions predicted to occur in the interface are likewise in agreement with earlier observations on Ag-Ab complexes. The epitope is formed by amino acids that are conserved among major allergens from related species within the Fagales order. In combination with a surprisingly high inhibitory capacity of BV16 with respect to allergic patients' serum IgE binding to Bet v 1, these observations provide experimental support for the proposal of dominant IgE epitopes located in the conserved surface areas. This model will facilitate the development of new and safer vaccines for allergen immunotherapy in the form of mutated allergens.

Submonolayer growth with decorated island edges

We study the dynamics of island nucleation in the presence of adsorbates using kinetic Monte Carlo simulations of a two-species growth model. Adatoms (A atoms) and impurities (B atoms) are codeposited, diffuse and aggregate subject to attractive AA and AB interactions. Activated exchange of adatoms with impurities is identified as the key process to maintain decoration of island edges by impurities during growth. While the presence of impurities strongly increases the island density, a change in the scaling of island density with flux, predicted by a rate equation theory for attachment-limited growth [D. Kandel, Phys. Rev. Lett. 78 (1997) 499], is not observed. We argue that, within the present model, even completely covered island edges do not provide efficient barriers to attachment.

An Essential Role for Antibody in Neutrophil and Eosinophil Recruitment to the Cornea: B Cell-Deficient (μMT) Mice Fail to Develop Th2-Dependent, Helminth-Mediated Keratitis

Abstract Invasion of the corneal stroma by neutrophils and eosinophils and subsequent degranulation disrupts corneal clarity and can result in permanent loss of vision. In the current study, we used a model of helminth-induced inflammation to demonstrate a novel role for Ab in mediating recruitment of these inflammatory cells to the central cornea. C57BL/6 and B cell-deficient (μMT) mice were immunized s.c. and injected intrastromally with Ags from the parasitic helminth Onchocerca volvulus (which causes river blindness). C57BL/6 mice developed pronounced corneal opacification, which was associated with an Ag-specific IL-5 response and peripheral eosinophilia, temporal recruitment of neutrophils and eosinophils from the limbal vessels to the peripheral cornea and subsequent migration to the central cornea. In contrast, the corneas of μMT mice failed to develop keratitis after intrastromal injection of parasite Ags unless Ags were injected with immune sera. Eosinophils were recruited from the limbal vessels to the peripheral cornea in μMT mice, but failed to migrate to the central cornea, whereas neutrophil recruitment was impaired at both stages. With the exception of IL-5, T cell responses and peripheral eosinophils were not significantly different between C57BL/6 and μMT mice. Taken together, these findings not only demonstrate that Ab is required for the development of keratitis, but also show that recruitment of neutrophils to the cornea is Ab-dependent, whereas eosinophil migration is only partially dependent upon Ab interactions.

Dynamics of irregular copolymers.

Reptation dynamics of AB copolymers with irregular chemical structure are considered theoretically. It is shown that interactions between A and B monomers could result in a significant slowdown of copolymer dynamics in the disordered (macroscopically homogeneous) state. The dynamical copolymer length N* showing the crossover to the strongly retarded dynamics is calculated. It is shown that contour-length fluctuations (internal reptation modes) give rise to a strong reduction of the slowdown effect and to a strong increase of N* which becomes unrealistically high in the case of a genuinely random chemical structure. The following scaling dependence of N* is predicted for irregular block copolymers: N* proportional, variant delta(-8)chi(-8)n(-8)(0)N(3)(e), where delta is the degree of block polydispersity, chi the Flory AB interaction parameter, and n(0) the mean block length. The strongest dynamical effect of AB interactions is predicted for correlated random copolymers near the critical point related to the formation of microdomain superstructures.

Immunosensors based on supported lipid membranes, protein films and liposomes modified by antibodies

The comparison of physical properties, sensitivity and reproducibility of various detection parameters of several newly developed affinity biosensors for determination of human IgE and herbicide 2,4-D is presented. The protein film based biosensors were composed of antibody (swine anti-human IgE (Q-SwaHU/IgE) or monoclonal antibody (MAb) against herbicide 2,4-D) attached to thin gold support through cysteamine or cysteamine–bovine serum albumin. The antigen (Ag)–antibody (Ab) interaction was detected by measurement of conductivity. The detection limit for human IgE was ∼1 nM, however, the sign of response depends on the method of antibody attachment to the gold support. This type of design and detection method was not appropriate for detecting small molecules, like 2,4-D. In the case of metal (stainless steel or gold) supported lipid films (s-BLM), the sensor was constructed by means of binding of avidin-modified antibody to s-BLM contained biotinylated phospholipids. Measurement of electrical capacitance, C, and elasticity modulus in direction perpendicular to the membrane plane, E ⊥, allowed to detect the Ag–Ab reaction, that resulted in decrease of C and increase of E ⊥. Most reproducible results have been obtained with lipid films supported on thin gold layers with detection limit of determination of 2,4-D∼1 μM. The best reproducibility and sensitivity (0.1–1 nM) have been obtained in liposome immunoassay. The Ag–Ab reaction was monitored by means of measurement the changes of ultrasound velocity in liposome suspension.

The effects of occupational therapy with sensory integration emphasis on preschool-age children with autism.

Using single-subject research design, the effects of an occupational therapy intervention emphasizing sensory integration with five preschool children with autism were examined. In the AB design, nonengagement, mastery play, and interaction were measured, using videotape clips of each child's free play in the preschool. Following a 3-week baseline, an occupational therapist provided one-on-one sessions and consultation to teachers for 10 weeks. When baseline and intervention phases were compared, four children demonstrated decreased frequency of nonengaged behavior, and three demonstrated increased frequency of mastery (goal-directed) play. Improvements in frequency of interaction were minimal. The results support descriptions in the literature regarding the behavioral changes that children with autism can make when participating in intervention using a sensory integration approach.

J/ ψ production and suppression

In recent years, the J/ ψ signal in heavy-ion collisions at the CERN SPS has attracted much attention as a potential quark–gluon plasma signature. This review discusses the prediction of J/ ψ suppression and the systematics of this signature as a function of the transverse energy of the collision and the transverse momentum of the J/ ψ. Since J/ ψ suppression was already present in hadron–nucleus interactions, hadronic models of absorption by nucleons and scattering with comoving secondaries soon emerged. After a discussion of J/ ψ and other related production data in pp, pA , and AB interactions, hadronic suppression models are introduced and compared with the CERN SPS data. Plasma model predictions are also given for CERN SPS collisions as well as for the future RHIC and LHC heavy-ion colliders.

Statics and Dynamics of Symmetric Diblock Copolymers: A Molecular Dynamics Study

Extensive molecular dynamics simulations are carried out to study static and dynamic properties of symmetric diblock copolymer melts, both in the disordered and in the lamellar phases. The lamellar phase is constructed using the natural lamellar spacing, determined from constant pressure simulations. The non-Gaussian character of the chains in the disordered phase is demonstrated and quantified. In the lamellar phase, the density profile of the separate blocks, as well as the interface thickness are determined as a function of chain length N and AB interaction parameter e, and compared with experiments and other theoretical results. Single chain and single block form factors indicate that the chains in the lamellar phase are distorted into stick-like objects. Our results in the disordered phase show a stronger dependence of the diffusion constant on the chain length than observed in previous simulations. Diffusion within the lamellar plane for systems with chains of length N ≤ 100 monomers is found to be ...

RAGE-AB Interactions in the Pathophysiology of Alzheimer's Disease

RAGE is a cell surface molecule primarily identified for its capacity to bind advanced glycation end-products and amphoterin. Immunocytochemical studies demonstrated that in Alzheimer's Disease (AD) the expression of RAGE is elevated in neurons close to neuritic plaque beta-amyloid (AB) deposits and in the cells of AB containing vessels. Cross-linking of surface bound AB 1-40 to endothelial cells, yielded a band of 50 kDa identified as RAGE. Using the soluble extracellular domain of recombinant human RAGE, we found that AB binds to RAGE with a Kd = 57 ± 14 nM, a value close to those found for mouse brain endothelial cells and rat cortical neurons. The interaction of AB with RAGE in neuronal, endothelial, and RAGE-transfected COS-1 cells induced oxidative stress, as assessed by the TBARS and MTT assays. ELISA demonstrated a 2.5 times increase of RAGE in AD over control brains. Activated microglia also showed elevated expression of RAGE. In the BV-2 microglial cell line, RAGE bound AB in dose dependent manner with a Kd of 25 ± 9 nM. Soluble AB induced the migration of microglia along a concentration gradient, while immobilized AB arrested this migration. AB-RAGE interaction also activated NF-kB, resulting in neuronal up-regulation of macrophage-colony stimulating factor (M-CSF) which also induced microglial migration. Taken together, our data suggest that RAGE-AB interactions play an important role in the pathophysiology of Alzheimer's Disease.

Human IgG2 variants of chimeric anti-CD3 are nonmitogenic to T cells.

The mouse anti-human CD3 mAb OKT3 is a potent immunosuppressive agent used for the treatment of acute transplant rejection. OKT3 therapy is associated with acute toxicity resulting from in vivo T cell activation and systemic cytokine release, and a human anti-mouse Ab response. T cell activation is thought to be triggered by CD3 cross-linking mediated by the Abs bridging T cells and Fc receptor-bearing cells. Recent studies in a mouse model indicate that anti-mouse CD3 Abs with low affinity for Fc receptors can achieve immunosuppression without T cell activation, toxicity, or an anti-Ab response. To obtain an analogous Ab to improve the current anti-human CD3 therapy, a humanized Ab with low affinity for Fc receptors is needed. In this study, we introduced mutations into the upper CH2 region of IgG2 and expressed the altered Fc as chimeric OKT3 Abs. Compared with chimeric OKT3 IgG1, IgG2, IgG3, and IgG4, the IgG2 mutants were less mitogenic to T cells, and they did not induce the release of TNF-alpha, IFN-gamma, or IL-2. In parallel, we observed no functional interaction of the IgG2 mutant Abs with K562 cells, which express the IgG2-binding Fc receptor on their surface. Despite no measurable T cell activation, the mutant Abs could still modulate the CD3 complex. When coupled to a humanized anti-CD3, the IgG2 variant may provide a drug with less acute toxicity and immunogenicity, but may still retain potent immunosuppressive properties.

Redundant expression but selective utilization of nuclear factor of activated T cells family members.

Nuclear factor of activated T cells (NF-AT) complexes regulate the induction of many early T cell activation molecules. Four related proteins can function as the cytoplasmic subunit of NF-AT, and their overlapping expression patterns and the mild phenotype of the NF-ATp null mice suggest that they may be functionally redundant. We characterized the distribution and activation of cytoplasmic NF-AT proteins in mature lymphocytes and found that NF-ATc, NF-ATp, and NF-AT4/x/c3 are co-expressed and co-regulated in mature T and B cells. Each protein forms independent DNA binding complexes, and at physiologic concentrations, NF-ATc and NF-ATp complexes out-compete NF-AT4/x/c3 for occupancy of NF-AT sites from the IL-2, IL-3/granulocyte-macrophage CSF, IL-4, and CD40 ligand genes. This predicts heavily redundant immune regulatory functions of NF-ATp and NF-ATc, but distinct activities for NF-AT4/x/c3. Additionally, Ab interaction with NF-ATp induces high affinity NF-kappaB site interaction, suggesting that nuclear partners may dramatically vary the specificity of the NF-AT family.

A quenching fluoroimmunoassay for analysis of the pesticide propazine in an apolar organic solvent, reverse micelles of AOT inn-octane: Effect of the micellar matrix and labeled antigen structure

A simple way of directly observing antigen-antibody binding in a reverse micellar system,n-octane containing reverse micelles of aerosol OT (AOT), using the hydrophobic pesticide propazine as antigen, is described. We observed two processes during fluorescein-labeled propazine (FP)-antibody (Ab) interaction in reverse micelles: (1) quenching of the fluorescence of FP after mixing of Ab and FP (due immune complex formation) and (2) restoration of FP fluorescence after addition of excess propazine to the immune complex formed. We found that the quenching efficiency depends on both the properties of the reverse micellar system (surfactant concentration, hydration degreeW 0 = [water]/[surfactant]) and the structure of the labeled antigen. A quenching fluoroimmunoassay of propazine both in apolar organic solvents and in water is developed. The method is homogeneous. The quenching time is 10–30 min, and the detection limit of propazine is 100 nM (20 Μg/L) in organic solvent and 10nM (2 Μg/L) in water. Propazine can be added to the reverse micellar system when dissolved in AOT/octane, or in an octane/chloroform mixture, or in chloroform. This makes possible the use of the analysis directly for pesticide extracts in nonpolar organic solvents.

Molecular analysis of the antibody response to thyroglobulin and thyroid peroxidase.

In this review, we discuss the latest results concerning the molecular analysis of antibodies (Ab) directed toward thyroid autoantigens. In particular, we attempt to define patterns within the Ab repertoire that correlate best to their activities. Whilst a considerable amount is now known concerning the Ab response to thyroid peroxidase (TPO), there is still much we do not understand. We review evidence for the site of interaction of TPO-reactive Ab with native TPO. The Ab responses to thyroglobulin (Tg) and, in particular, the thyroid-stimulating hormone receptor (TSH-R), are much less well characterised. In this review, we focus on the molecular analysis of the Ab response to Tg and TPO, assessing the repertoire as it is currently known. In addition, we have tried to link this information with the analysis of the epitopes recognised by the various Ab. Finally, we discuss one of the more unusual features of the thyroid Ab repertoire, the use of D-D fusion at heavy chain junctions, and questions raised by our current state of knowledge, such as the role of Ab using germline V regions in antigen recognition.

Superantigen-like unconventional AgAb interaction: “positive” selection of rabbit appendix B-cells

Superantigen-like unconventional AgAb interaction: “positive” selection of rabbit appendix B-cells

Development of User-Friendly Computer Programs To Predict Solution Properties of Single and Mixed Surfactant Systems

We present a description of two user-friendly computer programs designed for the prediction of surfactant solution properties : (1) program PREDICT for single surfactants, and (2) program MIX for binary surfactant mixtures. We also present an overview of the molecular-thermodynamic theories on which these programs are based. Program PREDICT can be utilized to predict a broad spectrum of properties, including (i) the critical micellar concentration, (ii) the optimal micellar shape, size, and size distribution, (iii) phase behavior characteristics, and (iv) surface tensions. Program MIX can be utilized to predict the critical micellar concentration of a binary surfactant mixture, and the β AB interaction parameter characterizing the nonidealities of mixing at the micellar level. Examples of the predictive capabilities of programs PREDICT and MIX are presented and compared with available experimental data.

Influence of Conformational Asymmetries on Local Packing and Small-Angle Scattering in Athermal Diblock Copolymer Melts

Local structural correlations and small-angle scattering profiles arestudied using the polymer reference interaction site model (PRISM) integral equation theory for a series of structurally asymmetric diblock copolymer melts in the athermal limit. Motivated by recent experimental work on polyolefin and polydiene copolymers, we have chosen the block aspect ratios and the overall degree of copolymer asymmetry to be representative of those systems. The extent of nonrandom mixing in the fluid is quantified by calculating length scale dependent «effective compositions». These deviations from the bulk composition in the athermal fluid are compared to those of a fully interacting thermal system. The athermal contribution to nonrandom mixing, resulting from pure stiffness asymmetry, is quite small relative to the effects brought on by unfavorable enthalpic AB interactions. Nonetheless, deviations from random mixing in the athermal limit can lead to appreciable unfavorable enthalpic interactions between components within a thermodynamic perturbative scheme. Large departures from the widely assumed equality of the three partial collective structure factors are also found, suggesting that the extraction of a single χ parameter by fitting to a form which assumes incompressibility may in some cases be unreliable

Electrochemical transduction of an immunological reaction via s-BLMs

The application of cyclic voltammetry to biosensor devices based on solid supported bilayer lipid membranes (s-BLMs) is investigated. s-BLMs have been used for receptor-ligand contact interactions. This paper reports the electrochemical transduction of an antigen-antibody (AgAb) reaction by an s-BLM. The antigen (HBsAg) is incorporated into an s-BLM, which then can interact with its corresponding antibody (HBsAb) in the bathing solution. This AgAb interaction results in some remarkable changes in the electrical parameters (conductance, potential and capacitance) of s-BLMs. The magnitude of these changes are directly related to the concentration of the antibody in the bathing solution. The use of such an AgAb interaction via s-BLM as a transducing device for the detection of ligand-receptor contact reaction is proposed.

The role of antisperm antibodies in infertility

To discuss the role of antisperm antibodies (Ab) in infertility, including proposed mechanisms of antisperm Ab formation, assays for their detection, and treatments for immune-mediated infertility. Major studies in the published literature and data from The University of Texas Southwestern Medical Center, Division of Reproductive Endocrinology. Reports were reviewed that investigated the development and impact of alloimmunity and autoimmunity to spermatozoa in men and alloimmunity in women and the current methods of treatment for resultant subfertility. The exposure of spermatozoal antigens to the mucosal and systemic immune systems results in development of immunity to a multiplicity of spermatozoal epitopes. The evaluation of studies that examine the role of antisperm Ab in infertility is complicated by the lack of standardization of antisperm Ab assays and the difficulty in identifying those epitopes for antisperm Ab binding that are responsible for subfertility. Sperm-associated antisperm Ab and antisperm Ab in cervical mucus (CM) reduce sperm mobility and may also impair sperm-ovum interaction. The clinical significance of serum antisperm Ab in both men and women, however, is controversial. Current therapy for antisperm Ab associated infertility is empiric and largely unproven. Antisperm Ab on the sperm surface and in CM are implicated in the pathogenesis of a subset of patients with infertility. Further studies that determine the clinically relevant sites of antisperm Ab interaction will aid in directing the treatment of subfertility mediated by antisperm Ab.