Transformation of serum immunoglobulins and monoclonal antibodies into polyreactive immunoglobulins

It was shown that polyreactive properties of natural serum immunoglobulins or some monoclonal antibodies (i.e. their ability of binding to various antigenically dissimilar antigens) can be induced by the treatment with chaotropic ions (3.5 M KSCN, 5-30 min at 20 (C). Such transformation of monoclonal antibodies or serum immunoglobulins into polyreactive immunoglobulins (PRIG) seems to result of the changes in immunoglobulin conformation but is not from their unblocking. Interaction of PRIG with some antigens immobilized on the plate is strongly dependent on temperature unlike that of monospecific antibodies.     

Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor

Interleukin-1 (IL-1) defines two polypeptides, IL-1 alpha and IL-1 beta, that possess a wide spectrum of biological effects. Two natural antagonists of IL-1 action have been characterized: the IL-1 receptor antagonist (IL-1Ra) and a soluble form of the type II IL-1 receptor. Neutralizing autoantibodies to IL-1 alpha have also been detected in sera of healthy individuals and patients with autoimmune or inflammatory diseases. To characterize such antibodies molecularly, we attempted to generate B cell clones producing anti-IL-1 alpha human monoclonal antibody (HuMAb) by combining Epstein-Barr virus-immortalization and CD40-activation of B lymphocytes from individuals with circulating anti-IL-1 alpha. We describe herein the generation and properties of a natural IgG4/kappa anti-IL-1 alpha monoclonal autoantibody, HuMAb X3, that bound specifically to human IL-1 alpha, but not to IL-1 beta and IL-1Ra, with a high affinity (Kd = 1.2 x 10(-10)M). HuMAb X3 inhibited IL-1 alpha binding to IL-1 receptors and neutralized biological activities of both recombinant and natural forms of IL-1 alpha. A recombinant form of HuMAb X3 was found to display identical specific IL-1 alpha antagonism. The presence of somatic mutations within X3 variable regions suggests an antigen-driven affinity maturation. This study extends the demonstration of the presence of high affinity neutralizing anti-IL-1 alpha autoantibodies that can function as a third type of IL-1 antagonist.     

Effect of calcium and magnesium on binding of beta, gamma-methylene ATP to sarcoplasmic reticulum

Isolated sarcotubular membranes (SR) from skeletal muscle bound 3.7 nmol of beta, gamma-methylene [8-3H]ATP (AMP-PCP) per mg of membrane protein. Only one class of binding site was identified and the dissociation constant (K) for this site was 1.5 X 10(-5) M. Addition of 0.05% Triton X-100 increased the number of binding sites to 5.7 nmol/mg. ATP and ADP competitively inhibited AMP-PCP binding. The dissociation constants for ATP and ADP were 3.5 X 10(-5) M and 3.3 X 10(-6) M, respectively. Since this data was obtained in the presence of 5 mM EDTA, it was established that the sarcoplasmic reticulum has a high affinity for the metal free forms of ATP, ADP, and AMP-PCP. Magnesium concentrations in excess of 1 X 10(-4) M inhibited AMP-PCP binding. Lower concentrations of magnesium had little effect on AMP-PCP binding. The effect of calcium on AMP-PCP binding was biphasic. Calcium concentration between 1 X 10(-6) and 1 X 10(-4) M inhibited AMP-PCP binding. Inhibition was maximal at 1 X 10(-5) M. Calcium concentration above 1 X 10(-4) M facilitated analogue binding. Possible sites of magnesium and calcium actions are discussed.     

Brownian dynamics study of ion transport in the vestibule of membrane channels

The Immunoglobulin (Ig) binding capacity of Toxoplasma gondii tachyzoites was investigated using fluorescence flow-cytometry analysis. Polyclonal mouse, human and rat immunoglobulins without specific anti-Toxoplasma activity bound to parasites in a concentration-dependent manner, saturating them at circulating serum concentrations. The immunoglobulin class and subclass specificity of binding was investigated using irrelevant monoclonal antibodies. IgM, IgA and IgG reacted with the parasite membrane. The attachment of mouse IgM to the parasite surface was hampered by mouse IgG1, IgG2a, IgG2b and IgG3. The binding of mouse IgG was proportionally reduced with increasing concentrations of mouse monoclonal IgM. The binding of murine immunoglobulin was diminished when in presence of human IgG. Purified Fc- but not Fab portions of immunoglobulins, fixed to parasites. Using labelled calibrated beads, the Ig binding capacity of parasites was estimated to be 6900 +/- 500 sites per tachyzoite. The Kd of the T. gondii Fc Receptor (FcR) activity was determined at 1.4 +/- 0.1 microM (mean +/- SEM). Such FcR activity was reduced by phospholipase C, trypsin and pronase treatment of the parasites. These data show a low affinity FcR activity on T. gondii tachyzoites which recognizes Ig of different species and isotypes and is likely supported by a glycosyl-phosphatidylinositol (GPI)-anchored surface protein of the parasite.     

Binding characteristics of drugs to synthetic levodopa melanin

To define binding characteristics of drugs, levodopa melanin was prepared with the aid of mushroom tyrosinase. The binding of radiolabeled substances was studied with increasing concentrations of melanin in a fixed volume of potassium phosphate buffer (pH 7.4) at 37 degrees. The affinity and capacity of the drug binding were calculated according to Langmuir's adsorption isotherm. The affinity constant of various sympathomimetic amines such as (-)-amphetamine, (+)-amphetamine, (-)-ephedrine, (+/-)-octopamine, and (+)-norepinephrine ranged from 1.1 to 2.8 X 10(5) M-1. The binding capacity for the amines ranged from 1.4 to 3.2 X 10(-9) mole/mg. Although the capacity of (+/-)-cocaine for binding was similar to that of the amines, the affinity was slightly higher, 8.9 X 10(5) M-1. The binding of atropine to the synthetic melanin appeared to be a saturable process with the affinity and capacity values of 0.2 X 10(5) M-1 and 7.6 X 10(-9) mole/mg, respectively. Although the binding lacks stereoselectivity, the drugs vary in their capacity and affinity to bind with melanin. The observed differential pharmacological and toxicologic properties of drugs in the pigmented tissues may in part be related to their differential bi binding characteristics.     

Dietary excess of vitamin B-6 affects the concentrations of amino acids in the caudate nucleus and serum and the binding properties of serotonin receptors in the brain cortex of rats

Vitamin B-6 is a cofactor in many reactions of nitrogen metabolism. Deficiency alters tissue amino acid concentrations but effects of excess vitamin B-6 have not been well described. We fed female rats (218 g, 7 per group) 1 (control), 10, 100, 175 or 250x) the National Research Council recommended level of pyridoxine HCl (7 mg/kg) for 10 wk and measured serum amino acids, amino acids and neurotransmitters in brain regions and the binding properties of serotonin receptors in the cerebral cortex using a ketanserin binding assay. Rats were decapitated, and unheparinized blood was obtained. In the caudate nucleus, concentrations of glutamate, threonine, taurine, methionine, gamma-amino-butyric acid and the sum of the essential amino acids in groups 10X and 100X were approximately 130 to 180% of control levels (P < 0.05); groups 1X, 175X and 250X were not different. A similar pattern was seen in the serum for serine, glycine, aspartate and ornithine; the latter two amino acids increased to over 200% of control in group 100X. In the ketanserin binding assay, both the antagonist binding affinity and the maximal number of binding sites were higher for group 100X than for 1X, 175X and 250X, and were higher for 10X than for 1X. Norepinephrine in the raphe nucleus followed a similar biphasic pattern. Excess dietary pyridoxine affected brain and serum concentrations of some amino acids and binding properties of cortical serotonin receptors in a biphasic pattern over the range of concentrations fed in this study.     

Exploration of beta-turn scaffolding motifs as components of sialyl Le(X) mimetics and their relevance to P-selectin

Monocyclic and bicyclic lactam units representing beta-turn surrogates were incorporated into a sialyl Le(X) structure by replacement of the natural sugar components. Low micromolar activity was found in a new P-selectin binding assay.     

Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1

Binding of factor Xa to human umbilical vein endothelial cells (HUVEC) is contributed by effector cell protease receptor-1 (EPR-1). The structural requirements of this recognition were investigated. Factor Xa or catalytically inactive 5-dimethylaminonaphthalene-1sulfonyl (dansyl) Glu-Gly-Arg-(DEGR)-chloromethylketone-factor Xa bound indistinguishably to HUVEC and EPR-1 transfectants, and inhibited equally well the binding of 125I-factor Xa to these cells. Similarly, factor Xa active site inhibitors TAP or NAP5 did not reduce ligand binding to EPR-1. A factor X peptide duplicating the inter-EGF sequence Leu83-Phe84-Thr85-Arg86-Lys87-Leu88- (Gly) inhibited factor V/Va-independent prothrombin activation by HUVEC and blocked binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 approximately 20-40 microM). In contrast, none of the other factor X peptides tested or a control peptide with the inter-EGF sequence in scrambled order was effective. A recombinant chimeric molecule expressing the factor X sequence Leu83-Leu88 within a factor IX backbone inhibited binding of 125I-factor Xa to HUVEC and EPR-1 transfectants in a dose-dependent fashion, while recombinant factor IX or plasma IXa had no effect. An antibody generated against the factor X peptide 83-88, and designated JC15, inhibited 125I-factor Xa binding to HUVEC. The JC15 antibody bound to factor Xa and the recombinant IX/X83-88 chimera in a concentration dependent manner, while no specific reactivity with factors X or IXa was observed. Furthermore, binding of 125I-factor Xa to immobilized JC15 was inhibited by molar excess of unlabeled factor Xa, but not by comparable concentrations of factors X or IXa. These findings identify the inter-EGF sequence Leu83-Leu88 in factor Xa as a novel recognition site for EPR-1, and suggest its potential role as a protease activation-dependent neo-epitope. This interacting motif may help elucidate the contribution of factor Xa to cellular assembly of coagulation and vascular injury.     

The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein

The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP.     

Comparative study of the pathogenicity of Chlamydia for white mice of different ages

Binding of 125I-labelled triiodothyronine (T3) to rat liver and kidney cytosol was examined. In both organs a very similar binding protein was detected. The apparent equilibrium association constants amounted to 4.8 X 10(7) M-1 for the liver protein and to 1.8 X 10(7) M-1 for the kidney protein. In the two organs a high capacity for T3 binding was observed: maximal binding capacities of respectively 4.3 and 33.2 pmol per mg total cytosol protein. Displacement studies using thyroid hormone analogues showed that the binding exhibited similar specificity.     

Enrichment of immunoglobulin binding Plasmodium falciparum-infected erythrocytes using anti-immunoglobulin-coated magnetic beads

It has been shown that nonimmune, human immunoglobulins are bound to the surface of certain strains of Plasmodium falciparum-infected erythrocytes. We describe a novel way of enriching parasitized red blood cells (pRBC) for immunoglobulin binding/rosette formation using Dynabeads coated with antibodies raised against human immunoglobulins. Whole P. falciparum cultures were mixed with the precoated beads for approximately 120 min at room temperature, and the bound pRBC were isolated by magnetic force. The nonbound cell fraction contained ring-infected pRBC, immunoglobulin-negative, trophozoite-infected pRBC, and uninfected erythrocytes. A consistent elevation in the immunofluorescence and rosette formation rates of 100% and 86% respectively, was detected after the first enrichment and subcultivation. Protein A or G were also found to support binding of pRBC through surface-expressed immunoglobulin. The Dynabead technique is a novel way of enriching pRBC based on the immunoglobulin-binding capacity of the infected erythrocyte.     

Staphylococcal neutral phosphatase. A highly cationic molecule with binding properties for immunoglobulin

Staphylococcal neutral phosphatase (NPtase) was purified from two Staphylococcus aureus strains by sequential high salt extraction, ultracentrifugation and ion exchange chromatography. The enzyme showed maximum phosphatase activity at neutral pH, appeared as two bands in SDS-PAGE (31 and 32 kDa), and the isoelectric point was > 10. No close similarity between NPtase and other known bacterial proteins in respect of their N-terminal amino acid sequences was found. Purified NPtase bound rat and human polyclonal IgG [intact and F(ab')2 fragments], IgM, IgA, intact myeloma immunoglobulins, myeloma light chains, gamma heavy chain and, with a much lower affinity, Fc fragments. Furthermore, NPtase can bind serum albumin. Heparin, a highly negatively charged molecule, significantly inhibited NPtase binding to immunoglobulins and HSA, but did not inhibit the binding of specific antibodies to NPtase; this indicates that charge interactions are important. The newly characterized staphylococcal phosphatase with binding properties for immunoglobulin is an interesting bacterial protein that could be involved in post-infectious sequelae.     

Biphasic alterations in cardiac beta-adrenoceptor signal transduction mechanism due to oxyradicals

To assess the effects of oxyradicals on cardiac beta-adrenoceptors, G-proteins and adenylyl cyclase, rat heart membranes were incubated with xanthine (X) plus xanthine oxidase (XO) for different intervals. The basal as well as forskolin-, NaF-, 5'-guanylylimidodiphosphate and isoproterenol-stimulated adenylyl cyclase activities showed an increase at 10 min and a decrease at 30 min of incubation with X plus XO. Treatment of membranes with H2O2 also produced biphasic changes in adenylyl cyclase activities. The density of beta1-adrenoceptors was decreased when cardiac membranes were treated with X plus XO for 10 and 30 min whereas the affinity of beta1-adrenoceptors was increased after 10 min and reduced after 30 min of incubation. The beta2-adrenoceptors were not modified at 10 min whereas incubation of cardiac membranes with X plus XO for 30 min increased the affinity and decreased the density. Cholera toxin-stimulated adenylyl cyclase activity, cholera toxin-catalyzed ADP-ribosylation and stimulatory guanine nucleotide binding protein immunoreactivity in cardiac membranes were increased at 10 min and decreased at 30 min of incubation with X plus XO. However, the pertussis toxin-stimulated adenylyl cyclase activity, pertussis toxin-catalyzed ADP ribosylation and inhibitory guanine nucleotide binding protein immunoreactivity were not affected on treatment of membranes with X plus XO. Addition of superoxide dismutase plus catalase in the incubation medium prevented the X plus XO-induced alterations in adenylyl cyclase activities, stimulatory guanine nucleotide binding protein-related ADP-ribosylation and changes in the characteristics of beta-adrenoceptors except the increased affinity of beta1-adrenoceptors at 10 min of incubation. These data suggest that alterations in the beta1-adrenoceptor-linked stimulatory guanine nucleotide binding protein-adenylyl cyclase pathway due to X plus XO are biphasic in nature and these changes may likely be due to the formation of H2O2.     

Triiodothyronine binding to lymphocytes from euthyroid subjects and a patient with peripheral resistance to thyroid hormone

Triiodothyronine (T3) binding to Ficoll-Isopaque purified human lymphocytes was studied. During incubation of lymphocytes with [125I]T3 in a calcium-free medium at 37 degrees C, maximal uptake of T3 in nuclei occurrred after 2 h and declined after prolonged incubationd incubation . Incubation of lymphocytes with T3 concentrations ranging from 1 X 10(-11) TO 1 X 10(-9) mol/l and subsequent treatment with Triton X-100 to strip off [125I]T3 bound with low affinity was used for the estimation of affinity and capacity of nuclear T3 binding sites. The mean equilibrium affinity constant (Ka) estimated with the Scatchard method in 11 euthyroid healthy subjects was 4.5 X 10(9) l/mol, and the mean maximal binding capacity 25 X 10(-5) mol/100 mug DNA. In a female patient with peripheral resistance to thyroid hormone action, the estimated Ka was 3.5 X 10(9) l/mol and the number of T3 binding sites 37 X 10(-15) mol/100 mug DNA. Although not statistically different from the mean value in euthyroid subjects, this Ka value was outside the range of control values observed and was considered presumptive evidence that the nuclear T3 receptors in this patient have abnormally low affinity for its ligand. The nuclear T3 binding capacity in this patient was significantly increased.     

Radiolabeling of the rat P2X4 purinoceptor: evidence for allosteric interactions of purinoceptor antagonists and monovalent cations with P2X purinoceptors

The rat recombinant P2X4 purinoceptor was expressed in CHO-K1 cells, and binding studies were performed using the radioligand [35S]adenosine-5'-O-(3-thio)triphosphate ([35S]ATPgammaS). In 50 mM Tris/1 mM EDTA assay buffer, pH 7.4 at 4 degrees, [35S]ATPgammaS bound with high affinity to the P2X4 purinoceptor (KD = 0.13 nM, Bmax = 151 pmol/mg of protein). The purinoceptor agonists ATP and 2-methylthioadenosine triphosphate possessed nanomolar affinity for the P2X4 purinoceptor, whereas the antagonist suramin possessed much lower affinity (IC50 = 0.5 mM). Cibacron blue was more potent than suramin but produced a biphasic competition curve, whereas d-tubocurarine potentiated binding at concentrations in excess of 10 microM. The complex effects of cibacron blue and d-tubocurarine seemed to be due to an allosteric interaction with the P2X4 purinoceptor because these compounds affected radioligand dissociation, measured after isotopic dilution with unlabeled ATPgammaS. Cibacron blue (1-100 microM) and d-tubocurarine (0.1-1 mM) produced rapid (10 sec to 5 min) decreases or increases, respectively, in the level of [35S]ATPgammaS binding measured immediately after initiation of the dissociation reaction. However, the subsequent rates of radioligand dissociation were not markedly different from those measured in their absence. Monovalent cations produced similar affects on the P2X4 purinoceptor and, like d-tubocurarine, increased [35S]ATPgammaS binding. The actions of d-tubocurarine and sodium were not additive. The findings from this study indicate that [35S]ATPgammaS can be used to label the P2X4 purinoceptor and suggest that this binding can be enhanced by monovalent cations and d-tubocurarine and may be subject to negative allosteric modulation to varying degrees by different purinoceptor antagonists.     

High-dose intravenous immunoglobulins in the treatment of adolescent and adult HIV-infected hemophiliacs

In children infected with human immunodeficiency virus (HIV) placebo-controlled trials with intravenous immunoglobulins have resulted in a significant reduction in morbidity; however, the results of small trials in adolescents and adults have been inconsistent. In this study 17 HIV-infected hemophiliacs aged 9-30 years were treated with monthly intravenous immunoglobulins for an average of 32 months. At the end of the study, 8 years after the HIV infection, three patients (18%) had progressed to the acquired immunodeficiency syndrome (AIDS), and the average decrease in CD4 cells was 81 cells/microliter per year. The natural history of HIV infection in hemophiliacs in this age group shows a manifestation rate of AIDS between 11% and 26% 6-8 years after seroconversion and an average yearly decrease in CD4 lymphocytes of 68-110 cells/microliters. In conclusion, we observed no difference either in the manifestation rate of AIDS or in prognostic markers in this small cohort of HIV-infected hemophiliacs treated for more than 30% of their latency period with intravenous immunoglobulins compared to the well-documented natural history of HIV-infected hemophiliacs. However, none of the patients developed severe bacterial infections during the study period.     

The effector functions of immunoglobulins: implications for therapy

The effector functions of immunoglobulins of the G class (IgGs) are essential for their effective use in therapy. The functions that operate following complex formation with cognate antigen involve binding to C1q (to mediate complement fixation) and the Fc receptors, Fc gamma RI, II and III. Another class of functions that is independent of antigen binding encompasses the transfer of antibodies across the placenta and maintaining the levels in the serum. All effector functions of IgGs are conferred by sequences in the Fc region of antibodies, and this review discusses the localisation of the functions to specific amino acid residues. Such knowledge is of use for the further improvement of IgCs for therapy.