A new set of monoclonal antibodies against human Fc gamma RII (CD32) and Fc gamma RIII (CD16): characterization and use in various assays

Four mouse anti-human Fc gamma RII (CD32) (6C4, 2B2, 3D3, 93.4) (IgG1, kappa) and one anti-human Fc gamma RIII (CD16) (7.5.4) IgG1, kappa) MAbs were raised. An in vitro switch variant, 7.5.4Sw50 (IgG2b, kappa), was also derived from the 7.5.4 MAb. 6C4, 2B2, and 3D3 MAbs bind both Fc gamma RIIa and Fc gamma RIIb isoforms. Two of them (6C4 and 2B2 MAbs) allow a complete blockade of the binding of immune complexes to Fc gamma RII. All three MAbs immunoprecipitate the receptor and bind both its glycosylated and nonglycosylated forms. The fourth anti Fc gamma RII MAb, 93.4, directed against the intracellular region of Fc gamma RIIa1/2, allows its detection by Western blotting only when it is not phosphorylated. The 7.5.4 MAb binds both Fc gamma RIIIa and Fc gamma RIIIb, can be used in Western blotting and does not inhibit aggregated IgG binding. ELISA using IV.3 (anti-Fc gamma RIIa1/2)/6C4 and 3G8 (anti-Fc gamma RIIIa/b)/7.5.4Sw50 MAb pairs make it possible to detect soluble Fc gamma RIIa1/2 and Fc gamma RIII, with a sensitivity of 200 pg/mL and 1 ng/mL, respectively. Surface plasmon resonance analyses indicated that the KD of two of the three anti-Fc gamma RII and of the anti-Fc gamma RIII are in the same order of magnitude (6C4: 0.78 nM, 2B2: 0.28 nM, 7.5.4: 0.47 nM). The anti-Fc gamma RII 3D3 MAb exhibits an off-rate constant higher than the 6C4 and 2B2 MAbs and a KD of 2.19 nM.     

Monoclonal antibody to the type II Fc receptor for human IgG blocks potentiation of monocyte and neutrophil IgG-induced respiratory burst activation by aggregated C-reactive protein

The acute phase protein, CRP, when heat-aggregated (Agg-CRP), binds to human monocytes and neutrophils and potentiates the respiratory burst stimulated by heat-aggregated IgG (Agg-IgG). Earlier data from our laboratory and others have indicated that CRP binds to phagocytic cells at membrane sites associated with IgG Fc receptors. The present study utilized monoclonal antibodies (MAb) to determine whether the Agg-CRP potentiation of oxidative metabolism could be linked to activation through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Preincubation of monocytes with MAb 32.2, which recognizes an Fc gamma RI epitope distinct from its IgG binding site, had only a minimal (20%) inhibitory effect on Agg-IgG-induced luminol chemiluminescence (CL) and exerted no significant effect on its enhancement by Agg-CRP. MAb 10.1, which blocks IgG binding to Fc gamma RI, reduced Agg-IgG-induced monocyte CL by 40%, but did not alter the Agg-CRP-mediated enhancement. In contrast, exposure to MAb IV.3, which binds to Fc gamma RII on monocytes and neutrophils and blocks IgG binding to this receptor, resulted in a greater than 70%, inhibition of Agg-IgG-induced CL and also significantly suppressed the enhancement by Agg-CRP. MAb Leu-11b, which reacts with Fc gamma RIII on neutrophils, reduced Agg-IgG-induced CL by 70% but did not suppress the Agg-CRP potentiation. Preincubation of monocytes and neutrophils with anti-Leu-M1, anti-CR1, or anti-CR3 failed to block Agg-IgG-induced CL or its enhancement by Agg-CRP. Although the potentiating effect of Agg-CRP on Agg-IgG-elicited CL was blocked by MAb IV.3, this antibody failed to reduce binding of Agg-CRP to either monocytes or neutrophils. These results indicate that, although Agg-CRP does not bind to phagocytic cells at the IgG-binding determinant of Fc gamma RII, it alters Agg-IgG-induced cell activation through this receptor.     

Murine IgG1 complexes trigger immune effector functions predominantly via Fc gamma RIII (CD16)

Previously, we have demonstrated that phagocytosis of IgG1-coated particles by macrophages in vitro is impaired by deletion of Fc gamma RIII in mice, suggesting that IgG1 may interact preferentially with Fc gamma RIII. In the present study, the biologic relevance of this observation was addressed by triggering various effector functions of the immune system in Fc gamma RIII(-/-) mice, using panels of mAbs of different IgG subclasses. Both binding and phagocytosis of IgG1-coated sheep or human erythrocytes by Fc gamma RIII(-/-) macrophages in vitro were strongly impaired, indicating that the impaired ingestion of complexed IgG1 by Fc gamma RIII(-/-) macrophages is due to a defect in binding. An in vivo consequence of the defective phagocytosis was observed by resistance of Fc gamma RIII-deficient mice to experimental autoimmune hemolytic anemia, as shown by a lack of IgG1-mediated erythrophagocytosis in vivo by liver macrophages. Furthermore, trapping of soluble IgG1-containing immune complexes by follicular dendritic cells in mesenteric lymph nodes from Fc gamma RIII(-/-) mice was abolished. Whole blood from Fc gamma RIII(-/-) mice was unable to induce lysis of tumor cells in the presence of IgG1 antitumor Abs. Finally, IgG1 mAbs proved unable to mount a passive cutaneous anaphylaxis in Fc gamma RIII(-/-) mice. Together, these results demonstrate that IgG1 complexes, either in particulate or in soluble form, trigger in vitro and in vivo immune effector functions in mice predominantly via Fc gamma RIII.     

Anti-Fc gamma receptor III autoantibody is associated with soluble receptor in rheumatoid arthritis serum and synovial fluid

We sought to detect anti-Fc gamma receptor (Fc gamma R) autoantibodies and soluble Fc gamma R in the serum and synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to correlate these serological abnormalities to the polymorphonuclear cell (PMN) activation state. Paired samples of blood and SF were obtained from 33 RA patients as well as blood from 25 normal adults from SF from 20 non-RA patients. Anti-Fc gamma R autoantibodies were assessed by an enzyme-linked immunosorbent assay (ELISA) using recombinant human Fc gamma R as the substrate. Soluble Fc gamma RIII was determined by an ELISA based on the combination of two monoclonal antibodies (MAb). The mean fluorescence intensity (MFI) of complement receptor 1 (CD35) and 3 (CD11b) and Fc gamma RIII (CD16) was evaluated by flow cytometry on the membrane of PMN. IgM anti-Fc gamma RIII activity was present in seven RA sera and five SF, and IgG in eight RA sera and six SF. The average concentration of soluble Fc gamma RIII was 1.80 +/- 3.50 micrograms/ml in RA patients and 0.33 +/- 0.06 micrograms/ml in normal adults (P < 0.05). This was elevated in the SF of 15 RA, while normal in that of all the non-RA patients. There was an inverse correlation between the CD16 MFI on the PMN and the serum/SF soluble Fc gamma RIII level, whereas the density of CD35 and CD11b was markedly augmented. Anti-Fc gamma RIII activity exists in RA patients, associated with soluble Fc gamma RIII. PMN activation could be due to these autoantibodies and thereby obviate the clearance of immune complexes.     

Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins

A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo.     

Tyrosine-containing activation motif-dependent phagocytosis in mast cells

FcR capable of triggering cell activation share with BCR and TCR a conserved intracytoplasmic tyrosine-containing activation motif (TAM). Besides cell activation, these receptors trigger other biologic responses, such as endocytosis of soluble ligands. Murine mast cells express two types of FcR that, when aggregated by antibodies and multivalent Ag, trigger the release of inflammatory mediators and cytokines. These are high affinity receptors for IgE (Fc epsilon RI) and low affinity receptors for IgG (Fc gamma RIII). They comprise each an IgE- or IgG-binding alpha-subunit and two TAM-containing subunits that associate with both receptors: a beta-subunit and a homodimeric gamma-subunit that can associate also with the other subunits of the TCR. Herein, we focused on biologic activities triggered in mast cells via the TAM of the gamma-subunits. Using rat basophilic leukemia (RBL) cells stably transfected with cDNA-encoding murine Fc gamma RIII alpha, we found that murine Fc gamma RIII trigger the phagocytosis of antibody-coated erythrocytes. Using RBL transfectants expressing Fc gamma RIII with a deletion of the intracytoplasmic domain of Fc gamma RIII alpha or chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the intracytoplasmic domain of Fc gamma RIII alpha, we showed that intracytoplasmic sequences of Fc gamma RIII alpha are neither necessary nor sufficient for Fc gamma RIII to trigger phagocytosis. Using RBL transfectants expressing chimeric receptors having the extracellular and transmembrane domains of Fc gamma RII and the TAM-containing intracytoplasmic domain of murine Fc gamma RIII gamma, we demonstrated that intracytoplasmic sequences of Fc gamma RIII gamma are sufficient to trigger phagocytosis. Using RBL transfectants expressing the same Fc gamma RII-III gamma chimeras, in the TAM of which one, the other, or both tyrosine residues were mutated, we established that tyrosines in the TAM sequence are required for phagocytosis. Our results endow TAM gamma with previously unknown triggering capacities and Fc gamma RIII with new biologic properties.     

Monomeric IgG2 enhances Ig production and proliferation in human B cells

The effect of purified polyclonal human IgG subclasses on B-cell responses was studied using the human IgA-producing B-cell line GM-1056. IgG2 at concentrations of 0.01-1 microgram/mL enhanced both IgA production and proliferation, while IgG1, IgG3, and IgG4 each failed to do so at tested concentrations between 0.001 and 10 micrograms/mL. This enhancement was Fc gamma R mediated, since IgG2 Fc fragments enhanced IgA production and proliferation to the same extent as did the whole IgG2 molecule, whereas F(ab')2 fragments did not. However, in contrast to monomeric IgG2, aggregated IgG2, which was expected to bind Fc gamma RII on B cells, affected neither IgA production nor proliferation. Similarly, anti-CDw32 mAb (2E1, anti-Fc gamma RII), anti-CD 64 mAb (32.2 anti-Fc gamma RI), and anti-CD16 mAb (Leu 11a, anti-Fc gamma RIII) mAb each failed to stimulate GM-1056 cells, and more importantly did not block IgG2-induced stimulation. Of various cytokines tested, including IFN-alpha, IFN-gamma, IL-1 beta, IL-2, IL-3, IL-4, IL-5, and IL-6, IL-6 alone augmented IgG2-induced enhancement of IgA production and proliferation. Moreover, the IL-6 effect was lost following preabsorption with anti-IL-6 antibody but not following preabsorption with control antibody. IgG2 also enhanced Ig production and proliferation in tonsillar large activated B cells, while IgG1, IgG3 and IgG4 each failed to do so. In contrast, IgG2 had no effect on Ig production and proliferation in tonsillar small resting B cells or SAC-stimulated small B cells. IgG2-induced enhancement of Ig production and proliferation in large B cells was not blocked by 2E1, 32.2, or Leu 11a, while enhancement was augmented in a specific fashion by IL-6. These results indicate that monomeric IgG2 specifically enhances B cell responses via an Fc gamma R receptor distinct from Fc gamma RI, Fc gamma RII, and Fc gamma RIII, and that IL-6 may play a role in augmenting this response.     

Bispecific-armed, interferon gamma-primed macrophage-mediated phagocytosis of malignant non-Hodgkin's lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.     

Distinct intracytoplasmic sequences are required for endocytosis and phagocytosis via murine Fc gamma RII in mast cells

In the present work, we studied the phagocytic and endocytic properties of murine Fc gamma RII in mast cells. Mouse mast cells express high-affinity receptors for monomeric IgE and three low-affinity receptors for complexed IgG: Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RIII. In previous studies we showed that, when aggregated by multivalent ligands, murine Fc gamma RIII, but not Fc gamma RII, triggers the release of inflammatory mediators and cytokines by mast cells. Upon Fc gamma R aggregation, mast cells not only release intracellular materials, they also internalize particulate and soluble immune complexes. We compared the ability of the two Fc gamma RII isoforms to trigger phagocytosis and endocytosis in RBL-2H3 cells stably transfected with cDNAs encoding wild-type, deleted, and tyrosine mutant Fc gamma RIIb1 or Fc gamma RIIb2. We found that Fc gamma RIIb2, but not Fc gamma RIIb1, triggered both phagocytosis and endocytosis. We identified distinct intracytoplasmic sequences necessary for Fc gamma RIIb2-mediated endocytosis and phagocytosis respectively, and we observed that two tyrosine residues, located in each of these sequences, are critical for endocytosis and/or phagocytosis. Our data indicate that the two internalization pathways diverge as early as signal transduction.     

An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells

The structure of the carbohydrate of the 40-kD major outer membrane component of Chlamydia trachomatis and its role in defining infectivity of the organism were investigated. The oligosaccharides were released from the glycoprotein by N-glycanase digestion, coupled to a 2-aminopyridyl residue, and subjected to two-dimensional sugar mapping technique. The major fractions consisted of "high-mannose type" oligosaccharides containing 8-9 mannose residues. Bi- and tri-antennary "complex type" oligosaccharides having terminal galactose were detected as minor components. These oligosaccharides were N-linked and contained no sialic acid. This structural profile is consistent with our previous characterization based on lectin-binding and glycosidase digestion. Functional specificity of identified chlamydial oligosaccharides was analyzed using glycopeptides fractionated from ovalbumin and structurally defined oligosaccharides from other sources. The glycopeptide fraction having high-mannose type oligosaccharide, as compared to those having complex or hybrid-type, showed a stronger inhibitory effect on attachment and infectivity of chlamydial organisms to HeLa cells. Among high-mannose type oligosaccharides, the strongest inhibition was observed with mannose 8 as compared with mannose 6, 7, or 9. These results indicate that a specific high-mannose type oligosaccharide linked to the major outer membrane protein of C. trachomatis mediates attachment and infectivity of the organism to HeLa cells.     

Workshop on in vitro neutralization of Chlamydia trachomatis: summary of proceedings

A task force evaluated an in vitro antibody-mediated chlamydial neutralization assay for its utility as a method to assess functional correlates of antibody responses to Chlamydia trachomatis. Two monoclonal antibodies that recognize different major outer membrane protein (MOMP) epitopes for a C. trachomatis serovar B strain exhibit good in vitro neutralizing activity, with a maximum of 90% neutralization. Calculations based on the 50% neutralization point indicated that 100% neutralization could theoretically be achieved when only 10% of the MOMP molecules bound antibody. Monoclonal antibodies that recognized either a heterologous MOMP or the genus-specific chlamydial lipopolysaccharide did not produce neutralizing activity. The standardized assay will be useful to establish if in vitro neutralizing antibody responses are predictive of protective immunity and will aid in defining chlamydial antigens and epitopes that may be attractive vaccine candidates.     

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.     

Differential expression of CR3, Fc epsilon RII and Fc gamma RIII on polymorphonuclear leukocytes in gingival crevicular fluid

Polymorphonuclear leukocytes (PMNLs) are the most numerous cell population among the cellular infiltrates in gingival crevicular fluid (GCF) and play important roles in the host-defensive system in the gingival crevices. We determined the percentage of neutrophils, eosinophils and basophils in total PMNLs by light microscopic observation using Randolph-methylene blue staining, then assessed flow cytometric differences in the expression of CR3, Fc gamma RIII, Fc epsilon RII, LFA-1 alpha, and LFA-1 beta on PMNL in GCF and peripheral blood (PB) from 21 patients with adult periodontitis (AP) and 13 healthy donors. Percentages of basophils and eosinophils were higher in GCF than in PB. In both AP patients and healthy subjects, expression of CR3 and Fc epsilon RII was higher while Fc gamma RIII was lower in GCF than in PB. The statistical analysis showed that the expressions of Fc gamma RIII and Fc epsilon RII on GCF PMNLs were lower in AP patients than in healthy subjects. Expressions of LFA-1 alpha and beta on GCF were similar to those on PB PMNLs. PB PMNLs stimulated in vitro with Porphyromonas gingivalis culture supernatant and fMLP displayed an expression pattern of CR3, Fc gamma RIII and Fc epsilon RII on GCF PMNLs. However, C5a and IL-1 failed to induce changes in Fc gamma RIII and Fc epsilon RII. The results indicate that GCF neutrophils are activated, present enhanced adhesion and a decreased IgG-binding ability which would reflect that they are at the terminal stage of activation, and that GCF contains a larger eosinophil fraction than in PB. Moreover, these GCF eosinophils appear to be activated.     

A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection

We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a beta-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56 degreesC, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.     

Regulation of human natural cytotoxicity by IgG. IV. Association between binding of monomeric IgG to the Fc receptors on large granular lymphocytes and inhibition of natural killer (NK) cell activity

The regulation of human natural killer (NK) activity by IgG described previously by us depends on the ability of cytophilic molecules of monomeric IgG (mIgG) to inhibit the subsequent killing of NK-sensitive targets. Highly purified NK cells obtained from human peripheral blood are able to directly bind mIgG as well as antigen-complexed IgG through its Fc region. The demonstration that NK cells bear labile cytophilic IgG, a property which usually has been attributed to L cells, indicates that mIgG-induced inhibition of NK activity is mediated by direct interactions between the inhibitory ligand and cytotoxic effector cells. The Fc receptor (FcR) mediating downregulation of NK cytotoxicity appeared to be FcR gamma III, previously found to be selectively expressed on NK cells and granulocytes. In studies of unidirectional cross-inhibition of mIgG binding to NK cells by various anti-CD16 monoclonal antibodies, binding characteristics of mIgG or complexed IgG were similar. Thus, the FcR gamma III for mIgG appear to be indistinguishable from receptors responsible for binding of polymeric IgG on human NK cells. The negative regulation of NK activity by mIgG was not attributable to inhibition of conjugate formation between effector cells and K532 targets, but rather to inhibition of a post-binding event involved in killing of conjugated targets. The data presented suggest that the Fc gamma RIII on human NK cells can either mediate killing against IgG antibody-coated target cells or, upon interaction with cytophilic monomeric ligand in soluble form, induce inhibition of NK activity.     

Human immunodeficiency virus type 1 attachment to HeLa CD4 cells is CD4 independent and gp120 dependent and requires cell surface heparans

The binding of human immunodeficiency virus type 1 (HIV-1) (Hx10) virions to two different cell lines was analyzed by using a novel assay based on the detection, by anti-HLA-DR-specific antibodies, of HLA-DR+ virus binding to HLA-DR- cells. Virion attachment to the CD4+-T-cell line A3.01 was highly CD4 dependent in that it was potently inhibited by CD4 monoclonal antibodies (MAbs), and little virus binding to the CD4- sister A2.01 line was observed. By contrast, virion binding to HeLa cells expressing moderate or high levels of CD4 was equivalent to, or lower than, binding to wild-type CD4- HeLa cells. Moreover, several CD4 MAbs did not reduce, but enhanced, HIV-1 attachment to HeLa-CD4 cells. CD4 was required for infection of HeLa cells, however, demonstrating a postattachment role for this receptor. MAbs specific for the V2 and V3 loops and the CD4i epitope of gp120 strongly inhibited virion binding to HeLa-CD4 cells, whereas MAbs specific for the CD4bs and the 2G12 epitopes enhanced attachment. Despite this, all gp120- and gp41-specific MAbs tested neutralized infectivity on HeLa-CD4 cells. HIV-1 attachment to HeLa cells was only partially inhibited by MAbs specific for adhesion molecules present on the virus or target cells but was completely blocked by polyanions such as heparin, dextran sulfate, and pentosan sulfate. Treatment of HeLa-CD4 cells with heparinases completely eliminated HIV attachment and infection, strongly implicating cell surface heparans in the attachment process. CD4 dependence for HIV-1 attachment to target cells is thus highly cell line specific and may be replaced by other ligand-receptor interactions.     

Novel monoclonal antibodies to putative selectin carbohydrate ligands that inhibit selectin binding to myeloid cells

Four newly developed monoclonal antibodies (MAbs) are characterized using flowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloid cells was tested. These MAbs react only with myeloid cells. MAbs CI-1, BU60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta2) integrins, while HI247 and CSLEX1 do not. The epitopes require Lewis x [Galbeta1-4 (Fucalpha1-3)GlcNAc] based on reactivity with oligosaccharide-polyacrylamide-biotin or oligosaccharide-BSA conjugates. MAb HI247 recognizes a related structure, sialyl-Lewis x, NeuAcalpha2-3GaLbeta1-4(Fucalpha1-3)GlcNAc. The three MAbs against Lewis x show some minor differences in their reactivity such as recognizing their antigens on CD11/CD18 integrins after endo-beta-galactosidase treatment and recognizing free Lewis x. The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid abolishes the binding of these MAbs. The C-3 sialic acid is crucial for the binding of MAb HI247. Its replacement by sulphate or its cleavage by sialidase eliminates recognition by this MAb. MAbs HI247 and CSLEX-1 did not react in ELISA with immobilized CD11/CD18, suggesting that the majority of sialyl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose. Unexpectedly, MAb BU60 inhibited binding of P-selectin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three other defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not. MAb HI247 inhibited binding of both E- and P-selectin chimeras to these cell lines more effectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sialyl Lewis x and related oligosaccharides. Certain combinations of these anticarbohydrate MAbs had additive inhibitory effects on selectin binding, suggesting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.     

Reactivities of feline calicivirus field isolates with monoclonal antibodies detected by enzyme-linked immunosorbent assay

Reactivities of feline calicivirus (FCV) field isolates with monoclonal antibodies (MAbs) were examined by enzyme-linked immunosorbent assay (ELISA). The reactivities of the viruses in ELISA were different from our previous results using the neutralization tests (NT). Many isolates were positive in ELISA with MAbs which recognized neutralizing epitope 3B and/or 4. However, most were negative in NT in our previous study. After absorption of two FCV strains with host cells, the non-infectious virus fluid still reacted with MAb, which recognized epitope 3B and/or 4 in ELISA. These results indicated the possibility that neutralizing epitopes are expressed on non-infectious virus particles or exist as proteinaceous molecules in virus fluid.     

Sulfated polysaccharides and a synthetic sulfated polymer are potent inhibitors of Chlamydia trachomatis infectivity in vitro but lack protective efficacy in an in vivo murine model of chlamydial genital tract infection

Heparin, dextran sulfate, pentosan polysulfate, and a sulfated synthetic copolymer of acrylic acid and vinyl alcohol were shown to be potent inhibitors of Chlamydia trachomatis infectivity for cultured human epithelial cells. Despite their potent antichlamydial activity in vitro, neither heparin nor dextran sulfate was effective in inhibiting the infectivity of C. trachomatis in a murine model of chlamydial infection of the female genital tract.