Differences in affinity of anti-CD4 monoclonal antibodies predict their effects on syncytium induction by human immunodeficiency virus.

A panel of 20 anti-CD4 monoclonal antibodies (mAb) was ranked in terms of affinity, using an inhibition radioimmunoassay. The ability of these antibodies to inhibit the induction of syncytia by human immunodeficiency virus (HIV) and to prevent binding of the HIV envelope glycoprotein 120 (gp120) to CD4 was also measured. Syncytium inhibition correlated strongly with affinity (P less than 0.001) but only weakly with inhibition of gp120 binding (P = 0.038). Some antibodies partially blocked binding of gp120 to CD4 but did not inhibit syncytia, and some antibodies inhibited syncytia but only weakly blocked binding of gp120. These results suggest that the syncytium inhibition assay is highly affinity-dependent, and that epitopes on CD4 concerned with virus binding are distinct from those involved in syncytium formation.     

The interaction of CD4 with HIV-1 gp120

CD4 is an integral cell surface glycoprotein that is able to enhance T cell specific antigen responses when it interacts with its physiological ligand, class II major histocompatibility (MHC) molecules. In addition, CD4 is a specific cell-surface receptor for the human immunodeficiency virus-1 (HIV-1). Infection by HIV-1 is initiated by the binding of the envelope glycoprotein, gp120, to the first domain of CD4. The binding of CD4 to class II MHC is inhibited by gp120, one possible mechanism for immunosuppression in AIDS patients. In addition, the CD4/gp120 interaction may directly inhibit T cell function. Recently we have synthesized small molecules (CPFs) that specifically inhibit this interaction. CPFs bind to gp120 and prevent the binding of gp120 to CD4, and also inhibit the infectivity of HIV-1.     

A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5

HIV-1 infection is initiated by the interaction of the envelope glycoprotein gp120 with the cellular receptor CD4 that triggers conformational changes in gp120 necessary for subsequent interaction with a coreceptor CCR5 (or CXCR4). The CD4-induced (CD4i) conformation of gp120 can be mimicked by a full-length single chain (FLSC) protein consisting of gp120 linked with the D1D2 domains of CD4 by a 20-amino-acid linker. We have used this protein to establish a flow cytometry-based assay and an ELISA-based assay to identify inhibitors that block the binding of gp120 to CCR5. Both assays are specific for detecting the known CCR5 antagonist TAK-779, but the ELISA-based assay was more sensitive, simple, inexpensive, and rapid; thus, it can be adapted to high throughput screening (HTS). The ELISA-based method was validated with a diverse set of known antagonists, for example, TAK-779, AOP-RANTES, PSC-RANTES, and several mAbs.     

Autoradiographic localization and characterization of [125I]lysergic acid diethylamide binding to serotonin receptors in Aplysia

The sensitive serotonergic radioligand 2-[125I]lysergic acid diethylamide was used to study the distribution and pharmacological binding properties of serotonin receptors in Aplysia californica. The high specific activity of this radioligand allowed us to develop a methodology for the investigation of receptor binding properties and receptor distribution in a single ganglion. [125I]Lysergic acid diethylamide labels a population of high-affinity serotonergic sites (Kd = 0.41 nM) in Aplysia ganglia whose regional distribution matches that expected from previous electrophysiological and immunohistochemical studies. The properties of [125I]lysergic acid diethylamide binding sites in Aplysia are in general agreement with previous studies on [3H]lysergic acid diethylamide binding in this system but these sites differ from the serotonergic receptor subtypes described in the mammalian brain. Guanine nucleotides were shown to modulate agonist but not antagonist affinity for the [125I]lysergic acid diethylamide binding site in Aplysia, suggesting that this site is coupled to a G-protein. Images of serotonin receptor distribution in the Aplysia nervous system were obtained from autoradiograms of [125I]lysergic acid diethylamide binding. Serotonin receptors in ganglia tissue sections are located primarily within the neuropil. In addition, a subset of neuronal soma are specifically labeled by [125I]lysergic acid diethylamide. These studies indicate that [125I]lysergic acid diethylamide binds to sites in the Aplysia nervous system which display a regional distribution, pharmacological binding properties and evidence of coupling to a G-protein consistent with labeling of a subset of functional serotonin receptors. In addition, the techniques used in this investigation provide a general approach for rapidly characterizing the pharmacological properties and anatomical distribution of receptor binding sites in single invertebrate ganglia. Individual neurons containing these receptor subtypes can be identified by these methods and correlated with physiological responses in the same cell.     

Modulation of CD4 lateral interaction with lymphocyte surface molecules induced by HIV-1 gp120

CD4, a lymphocyte surface glycoprotein, serves as co-receptor for antigen with the T cell receptor (TCR). It is also the lymphocyte receptor for HIV by binding the gp120 viral envelope protein. Interaction of gp120 with CD4 is crucial for viral infection, but is not sufficient to allow viral entry into cells. Recombinant gp120 alters CD4⁺ T cell responsiveness to activation stimuli. To express its co-receptor function fully, CD4 must be laterally associated with the TCR and CD45 to form multi-receptor complexes competent to transduce potent activation signals. Here, we examine the possibility that gp120/CD4 binding alters lateral associations of CD4 with other lymphocyte surface molecules, and that assembly of abnormal multi-molecular complexes is involved in the gp120-induced CD4⁺ T cell dysfunction and in viral entry. In the absence of gp120, CD4 displayed high association with CD3, CD5, CD45RC, CD25, CD28, CD44, and CD53; weak association with CD2, CD38, CD45RB, CD62L, and CD26; and no association with CD45RA, CD45RO, CD11b, CD11a, CD54, CD7, CD48, CD98, CD59, CD55, HLA class I and class II molecules. Treatment with gp120 significantly increased CD4 association with CD3, CD45RA, CD45RB, CD59, CD38, CD26, and HLA class I, and decreased that with CD45RC. Specificity of these results were assessed at various levels. First, gp120 did not influence lateral associations displayed by other molecules, such as HLA class II. Second, the Leu3 mAb, which binds CD4 on a site overlapping the gp120 binding site, did not elicit the same CD4 lateral associations as gp120, and finally, a direct gp120/CD4 interaction was needed to induce the lateral associations, as shown by the observation that blocking the gp120/CD4 binding by the Leu3 mAb inhibited the gp120-induced associations. These results can be interpreted in several ways. gp120/CD4 interaction could trigger an inside-out signal responsible for the associations, or gp120 could induce steric modifications of CD4 that increase its affinity for the associating molecules. Alternatively, these molecules may interact directly with gp120, bridging them with CD4. It is also possible that the associations may be mediated by additional components, interacting with both gp120 and the associating surface molecule. The last hypothesis is likely for CD59, whose gp120-induced association with CD4 required the presence of serum in the co-capping assay. Since both CD59 and gp120 bind complement, the observed association could be mediated by complement components.     

Pharmacological evidence for complex and multiple site interaction of CXCR4 with SDF-1alpha: implications for development of selective CXCR4 antagonists

The C-X-C chemokine SDF-1 and its receptor CXCR4, mediate a pivotal role in the pathophysiology of HIV-1 infection and vascular inflammatory diseases. In this study, we investigated the pharmacological properties of SDF-1alpha interaction with CXCR4 in human leukemia cell lines. Our data, based on [125I]-SDF-1alpha radioligand binding, SDF-1alpha-induced [35S]-GTPgammaS binding and use of specific CXCR4 antagonist AMD3100 reveals the complex nature of SDF-1alpha-CXCR4 interaction. Firstly, homologous competition with cold SDF-1alpha revealed a bimodal ligand displacement curve and secondly, although AMD3100 inhibited both SDF-1alpha-mediated chemotaxis (IC(50)=4.7 nM) and [35S]-GTPgammaS binding (IC(50)=7.4 nM) with high affinity, it was intriguingly up to 3000-fold less potent (IC(50)=15.2 microM) in the radioligand binding assay. These results provide pharmacological evidence for the recently described two-site model for SDF-1alpha-CXCR4 interaction. Accordingly, inhibition of SDF-1alpha binding to one of the receptor sites is sufficient to antagonize function, without causing its complete displacement from the receptor. Furthermore, these findings have important implications in the development and evaluation of CXCR4-selective small molecule antagonists for therapeutic use.     

Short synthetic peptides derived from viral proteins compete with HIV gp120 for the binding to CD4 receptors

In the complex mechanism of adhesion, internalization, and infection of cells by human immunodeficiency virus (HIV) viral particles, a determinant role is played by the viral envelope glycoprotein gp120, which binds to CD4 receptors of T cells and monocytes. We tested the ability of a panel of 7- to 12-residue synthetic peptides, selected from the region 414-434 of the HIV-1 gp120, to inhibit the binding of the viral protein to CD4 receptors of cultured human lymphoid cells. The assay was based on the observation that the binding of gp120 to the receptors interferes with the binding of a specific anti-CD4 monoclonal antibody, as a result of the masking of the antibody epitope; thus, we tested whether preincubation of cells with the peptides before gp120 addition might restore the recognition of the CD4 molecule by the antibody. High expression of CD4 receptors was thus assumed as indication that the binding of the viral protein had been inhibited. Maximum activity was displayed by a 9-residue peptide located near the amino terminal end of the 414-434 fragment. In addition, several fragments deduced from other viral proteins, possessing partial amino acid sequence homology with the HIV gp120 fragment, exhibited a similar type of interaction with the CD4 receptor. All active peptides contain the Cys residue (position 423 of gp120). This residue is essential, although not sufficient, for inhibiting gp120 binding, as few other amino acid residues within the fragment play a complementary role in increasing or decreasing the inhibitory ability.     

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction.

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.     

受体放射分析中标记配体质量的评价

受体的放射性配体结合分析是目前受体研究中常用的方法。其中，标记配体的质量十分重要。标记配体的质量主要有两个方面，即标记配体的纯度（结合力）及其与未标记配体的结合性能的一致性。目前，多数文献报道主雍以受体饱和结合试验的双倒数来测试标记配体的结合力，再以结合力校正标记配体量，得到活性的杯记配体量。在此基础上进行自身转换试验，测定标记配体比放射性及其与未标记配体结合性能的等效性。采用这些方法，作者对＾１     

The plasma protein which inhibits complement-mediated prevention of immune precipitation is an Fc binding protein

A glycoprotein (gp60) that inhibits complement-mediated prevention of immune precipitation (PIP) has been purified from normal serum. [125I]gp60 binds to IgG but not to IgA or IgM. The binding site has been shown to be localized on the Fc piece. The binding of radiolabelled gp60 to IgG has been analysed by direct binding and Scatchard, double-reciprocal and Hill plots. The mean affinity constant of gp60 for IgG is 1.56 x 10(8) l/mol and there appears to be a single class of binding sites for gp60 on IgG. Saturation was achieved when one molecule of gp60 was bound to each molecule of IgG. In competition inhibition assays, gp60 was shown to compete with C1q and IgM and IgG rheumatoid factors. The ability to inhibit C1q binding suggests that gp60 inhibits PIP by preventing binding and activation of C1. The possibility that gp60 is a fluid-phase Fc gamma receptor is discussed. See also the note added in proof.     

鸟类及鼠类的胸腺、腔上囊及脾脏褪黑素受体的鉴定及意义

应用放射配体结合法检测大鼠、小鼠、仓鼠、鸡、鸭、鸽子及鹌鹑的胸腺、腔上囊及脾脏的[~(125)I]褪黑素(M)特异结合位点。结果显示鸟类及鼠类免疫器官均存在[~(125)I]M特异结合位点,并具低结合容量、高亲和力特点;动力学研究表明具可饱和性及可逆性,符合受体的结合及解离过程;特异性研究显示对M及激动剂有高度特异性,符合特异结合位点的全部条件,亚细胞分布的研究表明以细胞核含量最高,线粒体次之,且该结合位点具年龄依赖性降低。结果证实免疫器官存在根黑素受体(MR),免疫组织是M作用的靶器官,M对免疫系统的调节作用是通过免疫组织上MR直接作用的结果。     

Depression of Rauscher leukemia virus envelope glycoprotein gp71 binding by lymphoid cells during leukemogenesis in mice.

The availability of membrane receptors for the 71,000-dalton envelope glycoprotein (gp71) of Rauscher murine leukemia virus on splenic and thymic cells from BALB/c mice during Rauscher murine leukemia virus-induced leukemogenesis was determined utilizing a radiolabeled gp71 binding assay. Shortly after infection, the relative cellular [125I]gp71 binding level decreased, first with splenic cells (at day 7 to 10 after infection) and later with thymic cells (at day 10 to 20 after infection). The dependency of the reduction of binding on the replication of the inoculated virus was demonstrated by regression analyses using cellular gp71 binding level as the dependent variable and infectious virus titer, as well as viral gp71 and p30 levels, of spleens and thymuses from infected mice as independent variables. With each independent variable, the reduction of gp71 binding for both cell types was highly dependent (P less than 0.01) on the level of virus detected in their respective organ. In the early stages of leukemogenesis, the [125I]gp71 binding level declined to approximately 20 to 30% of control values. During this period the rate of reduction of binding was very rapid and, in general was similar for both splenic and thymic cells. Further progression of the disease resulted in little or no further reduction in binding. The application of this technique to monitor host ecotropic virus synthesis and to study cell surface virus receptor control mechanisms in vivo is discussed.     

Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains

Human immunodeficiency virus (HIV-1) enters target cells by binding its gp120 exterior envelope glycoprotein to CD4 and one of the chemokine receptors, CCR5 or CXCR4. CD4-induced (CD4i) antibodies bind gp120 more efficiently after CD4 binding and block the interaction with the chemokine receptor. Examples of CD4i antibodies are limited, and the prototypes of the CD4i antibodies exhibit only weak neutralizing activity against primary, clinical HIV-1 isolates. Here we report the identification of a novel antibody, E51, that exhibits CD4-induced binding to gp120 and neutralizes primary HIV-1 more efficiently than the prototypic CD4i antibodies. The E51 antibody blocks the interaction of gp120-CD4 complexes with CCR5 and binds to a highly conserved, basic gp120 element composed of the beta 19-strand and surrounding structures. Thus, on primary HIV-1 isolates, this gp120 region, which has been previously implicated in chemokine receptor binding, is accessible to a subset of CD4i antibodies.     

Characterization of CD4 glycoprotein determinant-HIV envelope protein interactions: perspectives for analog and vaccine development

The CD4 surface determinant, previously associated as a phenotypic marker for helper/inducer subsets of T lymphocytes, has now been critically identified as the binding/entry protein for human immunodeficiency viruses (HIV). The human CD4 molecule is readily detectable on monocytes, T lymphocytes, and brain tissues. Soluble HIV (HTLV IIIB) envelope protein (gp120) binds native or recombinant CD4 with equal affinity estimated to be 4 to 8 nM kDa. All human tissue sources of CD4 bind radiolabeled gp120 to the same relative degree; however, the murine homologous protein, L3T4, does not bind the HIV envelope protein. Lack of sufficient recognition by the recombinant L3T4 molecule suggests divergence in the gp120-binding epitope. The binding of gp120 to CD4 is dependent upon intact sulfhydryl bonds within cysteine residues and glycosylation. Deglycosylated native gp120 is unable to bind CD4 under physiological conditions. Recombinant deglycosylated fragments cannot bind to the CD4 receptor, although they serve as immunogen for neutralizing antibody development. A number of synthetic peptides to putative critical domains of gp120 have been studied for their antagonism of native gp120 binding. Peptide T analogs or synthetic cogeners of Neuroleukin proposed to bind the CD4 determinant involved in gp120 binding had no competitive displacement of native gp120 binding as assessed by two independent methods that measure gp120 interaction with CD4. Recombinant C-terminal fragments, also containing other putative domains, did not displace native gp120 from CD4. Glycosylation appears to be critical in the maintenance of the structure of the binding domain of gp120. Native gp120 binding to CD4 is sufficient for the activation of cellular metabolism that alters target cell gene expression and differentiation, suggesting that the virus binding contributes to the activation of the host cell.     

Mutational Analysis of HIV-1 gp160-Mediated Receptor Interference: Intracellular Complex Formation

Formation of CD4–gp160 intracellular complexes represents an important mechanism leading to the induction of receptor interference. Previous studies have demonstrated that cells coexpressing gp160 and CD4 formed complexes of CD4 and gp160 which became blocked within the endoplasmic reticulum (ER), preventing CD4 from reaching the cell surface. In this report we have investigated the domains and residues of CD4 and gp160 involved in intracellular interaction. Accordingly, we have introduced mutations in both CD4 and gp160 at sites previously shown to disrupt CD4–gp120 interactions at the cell surface. Using a T7-vaccinia virus transient expression system, we expressed these gp160 and CD4 mutants in HeLa cells and analyzed their effects on intracellular complex formation and CD4 surface modulation. We observed that a number of gp160 mutants which failed to interact with CD4 at the cell surface also failed to bind and trap CD4 within the ER as expected. However, mutations at a critical residue, W427, did not abrogate intracellular CD4 binding. These gp160 mutants continued to interact with intracellular CD4 and inhibit CD4 transport to the cell surface, although gp120 produced from these mutants did not bind CD4 at the cell surface as expected. A number CD4 mutants also continued to form intracellular complexes with gp160, resulting in the loss of CD4 surface expression. Again, these CD4 mutants did not bind to gp120 at the cell surface, consistent with earlier reports. These results demonstrate that intracellular interactions between gp160 and CD4 in the ER may utilize different contact sites compared to those used during CD4 and gp120 binding at the cell surface. The data provide further evidence that the environment in which CD4 and the HIV-1 envelope glycoprotein interact can have a significant effect on their interaction.     

Glutamate carboxypeptidase II levels in rodent brain using [125I]DCIT quantitative autoradiography

The ability to visualize quantitatively glutamate carboxypeptidase II (GCPII) levels in vivo could advance our understanding of its function in health and disease. In the current study, we synthesized and evaluated a radiolabeled (iodine-125) analog of N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-S-3-iodo-L-tyrosine (DCIT), a potent antagonist of GCPII activity. We examined the regional distribution of [125I]DCIT binding in the rodent brain using quantitative autoradiography in order to confirm the validity of this radioligand as a marker of GCPII in the brain. The ultimate goal is to develop an imaging agent for assessing GCPII levels in the living brain. The specific binding of [125I]DCIT to rat brain followed a regional distribution consistent with previous studies describing regional brain GCPII gene expression and activity. We found a modest rostrocaudal gradient in which specific binding of [125I]DCIT to GCPII was lowest in cortical regions, with increasing levels of binding in midbrain structures and high levels of binding in hindbrain and brainstem. Autoradiography of [125I]DCIT in GCPII knockout and wild type mouse brain showed a gene-dose dependency confirming the selectivity of this radioligand for GCPII. We propose that [125I]DCIT is a selective radioligand that can be used to quantify brain GCPII levels in vitro using quantitative autoradiography.     

Inhibition of type 2,5'-deiodinase by tumor necrosis factor alpha, interleukin-6 and interferon gamma in human thyroid tissue.

The effects of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6) and interferon gamma (IFNgamma) were studied on the activity of type 2,5'-deiodinase and on the binding of [125I] T(4) to proteins in human thyroid cytosolic (supernatant) and membrane (pellet) fractions. The activity of thyroid type 2,5'-deiodinase was measured by iodothyronine outer ring deiodinase assay. The binding of [125I] T(4) to the proteins of thyroid cytosolic and membrane fractions was determined by autoradiography. The results showed that thyroid type 2,5'-deiodinase activity could be detected also in the cytosolic fraction, not only in the membrane. TNFalpha, IL-6 and IFNgamma could inhibit the type 2 deiodinase activity in vitro. The dose-dependent binding of [125I] T(4) to the proteins of 29, 66 and 200 kDa could be observed both in the cytosolic and membrane fractions. TNFalpha and IFNgamma inhibited the binding of [125I] T(4) to the two characteristic proteins of type 2,5'-deiodinase, to proteins of 29 and/or 200 kDa, both in the cytosolic and membrane fractions. We conclude that TNFalpha, IL-6 and IFNgamma can inhibit the activity of type 2,5'-deiodinase. Furthermore, TNFalpha and IFNgamma can still also decrease the binding of [125I] T(4) to the enzyme in vitro. It can be suggested that the increased level of these cytokines can be one of the reasons in the induction of the clinical symptoms in nonthyroidal illness associated with low levels of T(3).     

Distribution of [125I]omega-conotoxin GVIA and [3H]isradipine binding sites in the central nervous system of rats of different ages

Potential age-related changes in L- and N-type voltage-sensitive calcium channels (L- and N-VSCCs) were assessed by the in vitro binding of [3H]isradipine ([3H]ISR, 150 pM) and [125]omega-conotoxin GVIA ([125I]omega-CT, 4 pM) to membranes prepared from discrete central nervous system regions of 0.5-, 2-, and 18-month-old rats. The rank orders of [3H]ISR and [125I]omega-CT binding, although differing, indicated that the highest binding was in neocortex, corpus striatum, and hippocampus; radioligand binding was generally not affected by the variable of age. These results suggest that the nonidentical [3H]ISR and [125I]omega-CT binding sites are concentrated in those regions characterized by high densities of synaptic connections, and that these sites, as presumed components of L- and N-VSCCs, are relatively stable during the aging process.