Analysis of the expression of neutrophil-specific antigen NB1: characterization of neutrophils that react with but are not agglutinated by anti-NB1

The neutrophil-specific NB antigen system has been serologically characterized with human alloantisera. Two alleles, NB1 and NB2, have been described. NB1 is expressed on a subpopulation of peripheral blood neutrophils in 97 percent of healthy donors. Human alloantibodies have been used to identify the 58- to 64-kDa glycoprotein (GP) on which NB1 is located. NB1 can usually be detected by both a granulocyte immunofluorescence (GIF) assay and a granulocyte agglutination (GA) assay, but neutrophils from some donors have been found to react with anti-NB1 in GIF but not in GA assays. To determine if the latter neutrophils express NB1 and the corresponding 58- to 64-kDa GP, these neutrophils were probed with rabbit and human sera specific for NB1. First, the proportion of neutrophils that express NB1 was quantitated. Neutrophils from donors that typed as NB1-positive in both GA and GIF assays were analyzed by flow cytometry with antisera to NB1. Human and rabbit anti-NB1 reacted with 71 +/− 17 percent and 70 +/− 17 percent of neutrophils, respectively. There was no difference in the expression of NB1 in NB1-homozygous and NB1-heterozygous individuals. In contrast, significantly fewer neutrophils from four donors that typed as NB1- positive in GIF assay but not GA assay reacted with human (27 +/− 12%; p < 0.001) and rabbit (26 +/− 12%; p < 0.001) anti-NB1. When neutrophils from these same four donors were probed with rabbit and human anti-NB1 by immunoblotting and immunoprecipitation, the 58- to 64- kDa GP was identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinction of human immunodeficiency virus type 1 neutralization and infection enhancement by human monoclonal antibodies to glycoprotein 120.

There is increasing evidence that sera from HIV-1-infected individuals contain antibodies that enhance infection by HIV-1 in vitro. Previous work has demonstrated that complement receptors on T lymphoid cells and Fc receptors for IgG (Fc gamma R) on monocytic cells are required for enhanced infection by antibody-complexed HIV-1. Characterization of such infection-enhancing antibodies is essential because immunogenic epitopes which induce enhancing antibodies should be excluded from HIV-1 vaccines. This study was conducted to identify enhancing antibodies involved in Fc R-mediated enhancement of HIV-1 infection employing IgG human monoclonal antibodies (HMAbs) reactive against gp120 of HIV-1, which were produced by B cell lines derived from an HIV-1-infected individual. A potent neutralizing HMAb N70-1.5e did not enhance infection by HIV-1 (IIIB and MN strains), whereas HMAb N70-2.3a mediated enhancement of HIV-1 infection, but had little neutralizing activity. A competition radio immunoassay demonstrated that the two antibodies bind to distinct epitopes. These results indicated that enhancing and neutralizing antibodies can be induced by different epitopes on gp120, suggesting the potential for development of safe vaccines against HIV-1 by exclusion of immunogenic epitopes for enhancing antibodies. We made attempts to identify the epitope on gp120 that is recognized by the enhancing antibody N70-2.3a by using recombinant HIV-1 proteins and found that the antibody binds to a conformational site of nonvariable sequences in the carboxyl half (aa 272-509) of gp120.     

Monoclonal antibodies directed against the T-cell activation molecule CD137 (interleukin-A or 4-1BB) block human lymphocyte-mediated suppression of tumor xenografts in severe combined immunodeficient mice

A monoclonal antibody specific for the human analog of the murine T-cell activation molecule 4-1BB was generated and is shown here to react selectively with activated human CD4+ and CD8+ T lymphocytes. Treatment of these T cells in a one-way mixed lymphocyte culture with the anti-h4-1BB antibody enhanced the cell proliferation of the allostimulated lymphocytes. Previous studies in the mouse have shown that treatment of tumor-bearing mice with antibodies to 4-1BB augments anti-tumor immunity that is mediated by both CD4+ and CD8+ T cells. The authors consider the possibility that a similar approach may be efficacious for human cancer immunotherapy. This question was addressed by evaluating the effect of an anti-h4-1BB monoclonal antibody on human lymphocyte-mediated suppression of a human tumor xenograft in SCID mice. Mice treated with a control antibody and co-injected with the tumor and peripheral blood lymphocytes exhibited a lymphocyte dose-dependent suppression of tumor growth. In mice treated with the anti-h4-1BB antibody, the lymphocyte-mediated tumor suppression was completely eliminated and tumors grew progressively (as was observed in mice inoculated with tumors without lymphocytes). This monoclonal antibody specific for anti-h4-1BB, which augments the proliferation of allostimulated cells in vitro, blocks T-cell anti-tumor activity in vivo. These results suggest that although 4-1BB plays a role in the human peripheral blood lymphocyte-mediated suppression of tumor growth, antibodies to this molecule on human cells fail to stimulate anti-tumor activity, as was observed in tumor-bearing mice treated with an antibody to murine 4-1BB.     

Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1

The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa. The rev-specific fragment was isolated to immunize mice. Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA. Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.     

Polyclonal antibodies against gp185HER2 peptides: their putative role in the identification of a particular HER2 status in patients with breast cancer

The HER2 oncogene and its relative oncoprotein, gp185HER2, a transmembrane glycoprotein belonging to the epidermal growth factor receptor family, are overexpressed in a wide range of solid tumors including breast and ovarian cancer. In patients with breast cancer, both humoral and cell-mediated HER2 immune responses have been found as well as in some patients with gp185HER2 nonoverexpressing tumors. To establish whether peptide sequences identified as HLA-A2-restricted T-cell epitopes are expressed in breast tumor cell lines and tissues, we produced and characterized by different methodologic approaches polyclonal antibodies raised against four gp185HER2 peptides. Two of the antibodies recognized peptides eluted from the HLA-A2 groove of the mDAmB231 breast cancer cell line expressing a basal level of gp185HER2. Paraffin-embedded primary and metastatic breast tumors were specifically immunostained by all four reagents, thereby showing an overlapping reactivity. When this immunoreactivity was compared with that obtained using two different monoclonal antibodies, in 105 breast primary tumors and 36 corresponding lymph node metastases, we identified a subset of tumors that were negative with anti-gp185HER2 monoclonal antibodies and positive with the four antipeptide antibodies. Our novel observations provide in vivo evidence of the complexity involved in evaluating HER2 expression, and open a new path for understanding the biologic significance of HER2 status in breast tumors.     

Application of monoclonal antibodies to monitor the synthesis of a glycoprotein core of envelope glycoproteins of human immunodeficiency virus (HIV-1).

Using monoclonal antibodies 0.5 beta or G3-42, directed against V3 and C4 domains of glycoprotein 120 (gp120), we monitored the synthesis of oligomeric and monomeric forms of HIV-1 envelope glycoprotein 120 by flow cytometry or immunoprecipitation analysis in chronically infected MoIT-4 cells, cultured in the presence of tunicamycin. We observed that the inhibition of glycosylation by high concentrations of tunicamycin results in the reduction of an oligomeric gp120 on the surface of infected MoIT 4 cells as well as the decrease in the concentration of a monomeric form in the cytoplasm. Our studies revealed that the antibody 0.5 beta (exhibited higher sensitivity in the detection of gp120 than the antibody G3-42). We also observed that both antibodies did not recognise nonglycosylated precursor core envelope protein.     

Tuning luminescence of the fluorescent molecule 2-(2-hydroxyphenyl)-1H-benzimidazole via zeolitic imidazolate framework-8

Zeolitic imidazolate framework-8 (ZIF-8) is one of the most promising metal–organic frameworks because of its excellent high porosity, stability and geometrically well-defined structure. However, the application of ZIF-8 in the field of fluorescent molecular sensing has not been intensively explored. Our work demonstrates the versatility of ZIF-8 as a carrier material, which can be used for small molecule [2-(2-hydroxyphenyl)-1H-benzimidazole (HPBI)] capture and fluorescence enhancement. ZIF-8 displays luminescent behavior changes when combined with HPBI, as the emission peaks of ZIF-8 and HPBI are located in the same range for resonance enhancement of fluorescence. The results of the experiment indicate that the fluorescence enhancement effect will change in the presence of different concentrations of HPBI. We propose that the pore structure of ZIF-8 could provide an opportunity for the adsorption of HPBI molecules, and eventually the adsorption would saturate. The high porosity of ZIF-8 provides the path to HPBI aggregation or entrance into the ZIF-8 internal structure. Our results suggest that ZIF-8 may offer great promise for molecular fluorescence sensing.

ZIF-8 is regarded as a carrier to capture HPBI and eventually the adsorption of the pore structure would saturate, with altering the luminescent intensity. ZIF-8 may be used to sensitively detect nanoscale molecules via changes in fluorescence.     

Studies on the binding of an alloimmune and two murine monoclonal antibodies to the platelet glycoprotein IIb-IIIa complex receptor

The platelet-binding characteristics of three different antibodies that completely block adenosine diphosphate (ADP)-induced platelet fibrinogen binding and react with glycoproteins IIb, IIIa, or both, were studied. Two of the antibodies are murine monoclonal antibodies (10E5 and 7E3), and the third is an alloimmune antibody produced by a patient with Glanzmann's thrombasthenia who has received multiple transfusions (E.S.). The two monoclonals differ in that 7E3, but not 10E5, binds to dog platelets as well as to human platelets. In mutual competition studies, 7E3 did not interfere with 10E5 binding, indicating that both antibodies could bind to the complex simultaneously. E.S. IgG produced only minor inhibition of 10E5 binding, but nearly complete inhibition of 7E3 binding, suggesting that its epitope lies closer to the 7E3 epitope than the 10E5 epitope. Ethylenediaminetetraacetic acid (EDTA) pretreatment of intact platelets at 22 degrees C and pH 7.75 did not affect 10E5 binding, but significantly inhibited 7E3 binding. Similar treatment of intact or solubilized platelets at high pH and temperature produced splitting of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex and loss of both 10E5 and 7E3 binding. The 10E5 bound equally well to unactivated and activated platelets, and even rapid fixation of whole blood produced only a modest decrease in 10E5 binding. Preincubation of platelets with either E.S. IgG or 10E5 partially prevented EDTA from splitting the complex, and both monoclonal antibodies were able to bind to such complexes despite the EDTA treatment. These studies indicate that the binding of antibodies to several different epitopes on the GPIIb-IIIa complex can result in similar functional properties in blocking fibrinogen binding and platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies against viral determinants are not restricted to the K/D end of the major histocompatibility complex

Monoclonal antibodies against lymphocytic choriomeningitis virus (LCMV), a natural, high-replicating, noncytolytic pathogen in mice, were obtained from fusions between myeloma cells and lymphoid cells of mice of different H-2 haplotypes at various times (4-24 d) after infection. Supernatants from growing hybridomas were tested in a RIA, and approximately 15% of all supernatants were positive when tested for specificity on infected vs. uninfected cells of different haplotypes. Upon retesting for specific fluorescence, only some RIA+ supernatants exhibited specific surface staining of acetone-fixed infected cells or unfixed infected cells. In all these experiments and using various detection methods we could not find antibodies with any preference of recognition of viral antigen in conjunction with the H-2 haplotype of the responder mouse. The absence of H-2 restricted antibodies after a primary virus infection in vivo, whether assayed by RIA or surface immunofluorescence, suggests that antibodies obtained in other experiments using infected tumor cells for induction and in the RIA may not represent the general case.     

Adherence of neutrophils to cultured human microvascular endothelial cells. Stimulation by chemotactic peptides and lipid mediators and dependence upon the Mac-1, LFA-1, p150,95 glycoprotein family.

The process of neutrophil adhesion to and migration through the microvascular endothelium, an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractants. Although both chemotactic peptides and lipid mediators enhance neutrophil adherence in vitro and in vivo, the mechanism(s) involved in the interaction between circulating neutrophils and microvascular endothelial cells is still not completely understood. In a microtiter well adherence assay, the chemotactic peptides, FMLP and C5a, and the lipid mediators, leukotriene B4 (LTB4) and platelet activating factor (PAF), enhanced human neutrophil adherence to cultured human microvascular endothelial cells as well as to human umbilical vein endothelial cells in a dose-dependent manner with a rapid time course. This stimulated adhesive interaction between neutrophils and cultured human endothelial cells was dependent on the expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control neutrophils. All four mediators enhanced expression of the glycoprotein family on the surface of normal neutrophils as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the glycoprotein common beta subunit. In addition, TS1/18 inhibited up to 100% the adherence of normal neutrophils to endothelial cells stimulated by maximal concentrations of FMLP, C5a, LTB4, or PAF. Moreover, HL-60 cells, human promyelocytic leukemia cells, neither increased glycoprotein surface expression nor adherence in response to stimulation. Thus, peptide and lipid mediators of the acute inflammatory response appear to enhance adherence of circulating neutrophils to the microvascular endothelium by a mechanism dependent on expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface.     

Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils.

Two classes of adhesion molecules have well-defined roles in the attachment of unstimulated polymorphonuclear leukocytes (PMN) to cytokine-treated endothelial cells. Endothelial-leukocyte adhesion molecule 1 (ELAM-1) on endothelial cells interacts with specific carbohydrate residues on the PMN, and the leukocyte integrins (CD18 antigens) on PMN interact with intracellular adhesion molecule 1 and other structures on endothelium. Here we show that these two classes of molecules can act sequentially in an "adhesion cascade". Interaction of PMN with ELAM-1-bearing endothelial cells causes PMN to express enhanced adhesive activity of the integrin CR3 (CD11b/CD18). Expression of ELAM-1 on the cytokine-treated endothelium appears both necessary and sufficient for the stimulation of CR3 activity since blockade of ELAM-1 with mAbs prevents the activation of CR3 by cytokine-treated endothelium, and immobilized recombinant ELAM-1 activates CR3. The ability to activate CR3 is shared by chemattractants, suggesting that ELAM-1 may serve as a "tethered chemattractant." This hypothesis is strengthened by the observation that recombinant soluble ELAM-1 directs movement of PMN in chemotaxis chambers. These results suggest a mechanism by which multiple adhesive molecules may function together in diapedesis. ELAM-1 serves both as an adhesin and as a trigger that recruits the participation of additional adhesion molecules. Our results also suggest that ligands for adhesion molecules may also be "receptors" capable of generating intracellular signals.     

Study of the nature of A1- and A2-antigenic differences with use of monoclonal antibodies: a role of glycoprotein and glycolipid epitopes in their formation

The author used of A1- and A2-red blood cells to absorb Workshop-IV monoclonal reagents (anti-A1 2-24, 2-25, 2-27; anti-A 2-4, 2-10, 2-28; anti-H 2-71, 2-74) and anti-AHP protection, inhibited them with protein glycoconjugates (Apr), obtained by treating red blood cells with trypsin at pH 6.6 and by separating the supernatant on anion-exchange columns, and with lipid glycoconjugates (Alp), obtained by isolating glycosphingolipids from the trypsin-pretreated red blood cell membranes by the chloroform-methanol method and by subsequently performing anion-exchange gel chromatography. Judging from the results of the absorption using A1- and A2-red blood cells, the antigenic spectrum of A1 is more complete and exceeds that of A2, which permitted the use of the uniform test system of glycoconjugates from A1-red blood cells. Unlike monoclonal antibody's anti-A, the anti-A1-reagents were inhibited more slightly by glycolipid conjugates (other than those of the alkaline type (A1p-0), elution area pH 8.0-8.2), but more strongly by glycoprotein conjugates of the alkaline and moderately acid types (Apr-) and Apr-1 elution area pH 7.9 and 7.1) and they also reacted with the acid type of glycoprotein and glycolipid conjugates (Apr-3 and A1p-3 elution area pH 6.5-6.7). The use trypsin-treated A1-test-red blood cells dramatically decreased the titer of anti-A1-antibodes and failed to reveal the inhibitory activity of Apr-glycoconjugates. The difference in the inhibition of antibodies by glycoconjugates of various types, as well as the selectivity in the responsiveness of anti-A1-antibodies are attributable to the fact that A2-red blood cells show attenuation of not only glycosphingolipid epitopes of the alkaline types (A1p-0), but also glycoprotein epitopes of the acid type (Apr-3) in particular.     

Evaluation of an enzyme-linked immunosorbent assay kit (GTI PakPlus) for the detection of antibodies against human platelet antigens

Twenty-six serum samples from 24 patients were investigated for the presence of platelet-specific antibodies in a partly retrospective (n = 15) and partly prospective (n = 9) study. The sera contained either alloantibodies to human platelet antigens (HPA) (n = 23) or were from clinically suspected cases of fetomaternal alloimmune thrombocytopenia (FMAITP) in which platelet-specific antibodies had not been detected (n = 3). Three techniques were used to detect platelet antibodies: the platelet immunofluorescence test, the monoclonal antibody immobilization of platelet antigens (MAIPA) assay and a commercially available enzyme-linked immunosorbent assay--GTI PakPlus (GTI kit). Two alkaline phosphatase-conjugated antiglobulin reagents provided by the manufacturer were used in the GTI kit: an antihuman IgG/IgA/IgM (IgGAM) conjugate and an antihuman IgG conjugate. The GTI kit with the anti-IgGAM conjugate failed to detect eight antibody specificities in seven sera (anti-HPA-1a [n = 3], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1] and anti-HPA-5b [n = 3]). Greater signal-to-background ratios were achieved in the GTI kit with the anti-IgG conjugate but five antibody specificities (anti-HPA-1a [n = 1], anti-HPA-3a [n = 1], anti-HPA-3b [n = 1], anti-HPA-5b [n = 2]) remained undetectable. All the sera were detected by MAIPA assay and, furthermore, the MAIPA assay achieved the greatest signal-to-background ratio in the majority of sera tested. These findings re-emphasize the value of the MAIPA assay in reference laboratories and illustrate that the GTI kit may either fail to detect or incorrectly identify clinically significant HPA antibodies.     

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.