Method for detection of picornavirus capsid binders with soluble intercellular adhesion molecule 1.

A method utilizing immobilized major group rhinoviruses and biotinylated soluble intercellular adhesion molecule 1 has been developed for the detection of capsid binders. Binding measurements indicate a loss in relative affinity of biotinylated soluble intercellular adhesion molecule 1 for rhinoviruses by capsid binders. This method and a new class of capsid binders are discussed.

Monoclonal antibodies to lymphocyte function-associated antigen-1 inhibit invasion of human lymphoma and metastasis of murine lymphoma.

The leukocyte integrins are cell adhesion molecules which play pivotal roles in the development of a variety of immune responses including T-cell-mediated cytotoxicity, lymphocyte proliferation, macrophage presentation of antigen, and adhesion of leukocytes to vascular endothelium. The relevance of lymphocyte function-associated antigen-1 (LFA-1) to leukocyte malignancies is currently under examination in a number of laboratories. Here, we present evidence demonstrating that LFA-1 plays a role during the in vitro invasion of human endothelium by JY lymphoma cells and during in vivo metastasis of two distinct models of murine leukemia: P815 mastocytoma and EL4 lymphoma. When assayed in vitro, a murine anti-human LFA-1 (alpha subunit) monoclonal antibody (mAb) inhibits up to 80% of JY lymphoma cell invasion. When assayed in vivo, a rat anti-LFA-1 (alpha subunit) mAb significantly inhibited the development of experimental metastases, when administered concomitantly with either P815 or EL4 tumor cells. The leukocyte integrins, particularly LFA-1, may represent useful targets for the therapeutic modulation of metastasis.

Monoclonal antibody to MALA-2 (ICAM-1) reduces acute autoimmune nephritis in kdkd mice.

Hereditary tubulointerstitial nephritis is a prominent cause of renal failure in humans. A variety of animal models utilizing immunologically induced nephritis have been developed. The kdkd congenic variant of the CBA/Ca mouse has normal kidneys at birth but develops progressive, lethal autoimmune nephritis beginning at approximately Week 8. The destruction of renal tubular epithelium in mediated by a population of antigen-specific, H-2Kk-restricted, Lyt-2+, L3T4- T cells. The present experiments demonstrate that systemic treatment with anti-ICAM-1 monoclonal antibody reduces kidney disease in kdkd mice. Anti-ICAM-1 mab localizes to inflammatory sites in the kidney and effects a significant reduction in leukocyte infiltration. Concomitantly, urine protein levels of anti-ICAM-1-treated mice are significantly reduced. The use of anti-adhesion molecule monoclonal antibodies that alter leukocyte activity and/or trafficking may be useful therapies for certain autoimmune disorders.

In vitro studies of the antirhinovirus activity of soluble intercellular adhesion molecule-1.

We studied the in vitro antirhinovirus activity of a soluble form of intercellular adhesion molecule-1 (sICAM-1). sICAM-1 inhibited the cytopathic effect of 10 representative human rhinovirus (HRV) serotypes of the major receptor group with, 50% effective concentrations (EC50s) of 0.1 to 7.9 micrograms/ml. Cell type-dependent variation in the inhibitory activity of sICAM-1 was observed for two major receptor group serotypes in HeLa cells (EC50, greater than 32 micrograms/ml), and no inhibitory effect was observed for two serotypes which use different cell receptors. Yield reduction assays showed that sICAM-1 inhibited the replication of HRV serotype 39 (HRV-39) in human adenoid explants in a concentration-dependent manner. No direct inactivation of infectivity of HRV-39 (EC50, 0.5 microgram/ml) was observed after incubation with sICAM-1 (32 micrograms/ml) for up to 24 h. Single-cycle-of-replication experiments with the addition of sICAM-1 at 10 micrograms/ml at different times showed that the inhibitory effect occurs only when sICAM-1 is added within 30 min after infection. In experiments in which absorption was carried out at 4 degrees C and then a single cycle of replication incubation was carried out at 33 degrees C, it was found that sICAM-1 at 10 micrograms/ml was inhibitory only when it was present during the absorption period. Our data show that sICAM-1 is inhibitory for representative major receptor group serotypes of HRV in two cell lines and human respiratory epithelium, that the interaction of sICAM-1 with HRV is readily reversible by dilution, and that the inhibitory effect of sICAM-1 on virus replication is present early in the infection cycle.

Nonnucleoside inhibitors of HIV-1 reverse transcriptase: nevirapine as a prototype drug.

Nevirapine, a dipyridodiazepinone, is a highly specific inhibitor of HIV-1 reverse transcriptase (RT) which exhibits an IC50 = 84nM in enzyme assays and IC50 = 40nM against HIV-1 replication in cell culture. This nonnucleoside inhibitor acts noncompetitively with respect to nucleoside triphosphates, template and primer suggesting that nevirapine does not bind to the active site of RT. Studies employing an azido analogue of nevirapine as a photoaffinity probe indicated that one molecule of inhibitor is sufficient to inactivate one molecule of heterodimeric enzyme and demonstrated that only the p66 subunit of p66/p51 heterodimeric RT is covalently labeled by this probe. When subjected to trypic mapping, Tyr 181 and Tyr 188 were labeled with probe and consequently these aromatic residues are apparently near or actually within the RT binding site for nevirapine. The extent to which Tyr 181 and Tyr 188 participate/contribute to nevirapine binding was determined by making amino acid substitutions at these positions using the corresponding residues from HIV-2 RT which is not sensitive to nevirapine. A change at either position dramatically decreased the enzymes' sensitivity to nevirapine, as well as to TIBO derivative and Merck L-693,593, indicating that both Tyr 181 and 188 are crucial for inhibitor-enzyme interaction. Cell culture selection in the continued presence of nevirapine results in the appearance of resistant HIV-1, Tyr 181 to Cys, raising the concern that combination drug therapy will be required in the clinic.

Detection of major group rhinoviruses by soluble intercellular adhesion molecule-1 (sICAM-1).

Soluble intercellular adhesion molecule-1 (sICAM-1) was shown to be the receptor for the major subgroup of rhinoviruses. It was demonstrated that this molecule can inhibit the binding and subsequent infection of target cells by rhinoviruses belonging to the major but not the minor subgroup. The data reported now describe an ELISA-based system utilizing biotinylated sICAM-1 as a means of detecting rhinoviruses belonging to the major subgroup.

Reduction in the severity of graft-versus-host disease and increased survival in allogenic mice by treatment with monoclonal antibodies to cell adhesion antigens LFA-1 alpha and MALA-2.

Bone marrow transplantation is a therapeutic treatment for many life-threatening hematologic disorders, especially leukemia and certain immune deficiency diseases. However, acute graft-versus-host disease is often associated with bone marrow transplantation. In mice, allogeneic GVHD appears to be mediated by both host natural killer cells and donor T cells. In vitro and in vivo experiments demonstrate that treatment with either YN1/1.7 or M17/4.2 mabs is immunomodulatory and inhibits both the mixed lymphocyte reaction and natural killer cell activity. In addition, utilizing an allogeneic model of acute, lethal GVHD with C57B1/6 mice as donors and sublethally irradiated BDF1 mice as recipients, treatment of host mice with anti-LFA-1 alpha (M17/4.2) or anti-MALA-2 (YN1/1.7) mabs at a dose of 10 mg/kg/day for 10 days significantly reduced GVHD and enhanced survival. Mabs to lymphocyte adhesion molecules such as LFA-1 alpha and MALA-2 may provide a useful therapy for the treatment of GVHD.

Serum levels of circulating intercellular adhesion molecule 1 in human malignant melanoma.

Current reports have suggested a role for intracellular adhesion molecule 1 (ICAM-1) in the progression of human malignant melanoma and other cancers. Stage I, II, and III patients with histologically diagnosed malignant melanoma had significantly increased serum levels of circulating ICAM-1 (cICAM-1) and a striking increase in the incidence of positive sera. In Stage II and III patients, the level of cICAM-1 was inversely correlated with survival. Patients with elevated levels of serum cICAM-1 (greater than 2 SD units above control mean) had a significantly shorter mean survival. We suggest that elevated levels of serum cICAM-1 may be of diagnostic and prognostic importance in patients with malignant cutaneous melanoma.

Distribution of ICAM-1 within decidua and placenta and its gestational age-associated changes.

Intercellular adhesion molecule 1 (ICAM-1), a ligand of leukocyte function associated antigen 1 (LFA-1), is present on many cells, including monocyte/macrophages. ICAM-1 is considered to play an important role in the induction and maintenance of inflammatory responses by permitting leukocyte adhesion. Its expression is inducible on endothelial and epithelial cells exposed to various inflammatory cytokines (preceding expression of HLA-DR) and is maturation dependent in certain cell lines. The distribution of ICAM-1 in decidua and placenta was evaluated using peroxidase-antiperoxidase immunohistochemistry. In decidua of first and third trimesters, scattered ICAM-1-staining cells were observed. In placentas of first and third trimesters, all types of trophoblasts stained negatively for ICAM-1. Prior to 10 weeks of gestation, the villous stroma was uniformly ICAM-1 and HLA-DR unreactive. Beginning in the chorionic plate at approximately 10 weeks, scattered ICAM-1-positive stromal cells were observed, whereas stromal cells of the terminal villi revealed no ICAM-1. By 14-16 weeks, approximately 40-50% of the terminal villous stromal cells were ICAM-1 staining. This parallels the 40-50% of the villous stromal cells that share other immunohistochemical markers, such as EB-11, with monocyte/macrophages. The lack of functional maturation of the villous stromal macrophage may explain the rarity of chronic villitis early in gestation.

Inhibition of human immunodeficiency virus type 1 (HIV-1) replication by the dipyridodiazepinone BI-RG-587.

The dipyridodiazepinone human immunodeficiency virus type 1 (HIV-1)-specific reverse transcriptase (RT) inhibitor BI-RG-587 was tested for its ability to inhibit HIV-1 replication in both acutely and chronically infected cell lines. The ability of BI-RG-587 to inhibit steps in the virus replicative cycle other than reverse transcription was also assessed. BI-RG-587 was found to be a potent inhibitor of HIV-1 replication in acutely infected cells (50% inhibitory concentration [IC50] = 37.2 nM), and the sensitivity and kinetics of that inhibition was similar to the known RT inhibitor zidovudine (AZT). Even at 100x IC50, BI-RG-587 had no effect on gp120/CD4 interaction, syncytia formation, or envelope glycoprotein processing. In addition, no inhibition of viral replication or protein production was noted in a chronically infected cell line that produces viral products in an RT-independent manner. Finally, no inhibition of acute HIV-2 replication was noted, even with very high (2500x IC50 for HIV-1) concentrations of BI-RG-587. These results demonstrate that BI-RG-587 is a potent inhibitor of HIV-1 replication and that this inhibition occurs at the point of reverse transcription.

BI-RG-587 is active against zidovudine-resistant human immunodeficiency virus type 1 and synergistic with zidovudine.

A series of dipyridodiazepinones have been shown to be potent inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. The lead compound, BI-RG-587, had a 50% inhibitory concentration of 84 nM against HIV-1 reverse transcriptase activity. This compound reduced plaque formation of HIV-1 in HeLa cells expressing the CD4 receptor by 50% at 15 nM. BI-RG-587 at comparable concentrations inhibited the production of p24 antigen following the acute infection of CEM T-lymphoblastoid cells or primary human monocyte-derived macrophages with HIV-1. No inhibitory effects against HIV-2 or against three picornaviruses were detected. Zidovudine (3'-azido-3'-deoxythymidine [AZT])-susceptible and AZT-resistant isolates of HIV-1 were equally susceptible to BI-RG-587. AZT and BI-RG-587 exhibited synergistic inhibition of HIV-1BRU at all concentrations examined.

Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor.

A series of dipyridodiazepinones have been shown to be potent inhibitors of human immunodeficiency virus-1 (HIV-1) reverse transcriptase (RT). One compound, BI-RG-587, had a Ki of 200 nanomolar for inhibition of HIV-1 RT that was noncompetitive with respect to deoxyguanosine triphosphate. BI-RG-587 was specific for HIV-1 RT, having no effect on feline and simian RT or any mammalian DNA polymerases. BI-RG-587 inhibited HIV-1 replication in vitro as demonstrated by in situ hybridization, inhibition of protein p24 production, and the lack of syncytia formation in cultured human T cell lines and freshly isolated human peripheral blood lymphocytes. Cytotoxicity studies of BI-RG-587 on human cells showed a high therapeutic index (greater than 8000) in culture.

A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection.

Rhinoviruses belong to the picornavirus family and cause about 50% of common colds. Most rhinoviruses and some coxsackie viruses share a common receptor on human cells. The glycoprotein intercellular adhesion molecule-1 (ICAM-1) has recently been identified as the cellular receptor for the subgroup of rhinoviruses known as the major groups. ICAM-1 is a member of the immunoglobulin supergene family and is a ligand for lymphocyte function-associated antigen-1 (LFA-1); these ICAM-1/LFA-1 interactions are critical to many cell adhesion processes involved in the immunological response. Because anti-ICAM-1 antibodies can block binding of major-group rhinoviruses to cells, we considered that antagonism of virus-receptor interaction might be a way of preventing rhinovirus infection. We have constructed and purified a soluble form of the ICAM-1 molecule, which is normally membrane-bound, and demonstrated that it is a potent and specific inhibitor of rhinovirus infection.

Evaluation of zinc complexes on the replication of rhinovirus 2 in vitro.

The effect of zinc salts and complexes were evaluated on the replication of rhinovirus 2 in vitro. Zinc chloride inhibited the replication of rhinovirus 2 at concentrations between 3 and 12 micrograms/ml. Influenza virus was not affected. A number of zinc complexes were tested and compared to zinc chloride. The results indicated that the activity and toxicity of all zinc complexes in the rhinovirus cytopathogenic effect (CPE) assay were directly related to the amount of unbound zinc available.

Polypeptide 2A of human rhinovirus type 2: identification as a protease and characterization by mutational analysis.

Evidence is presented that the protein 2A of human rhinovirus serotype 2 (HRV2) is a protease. On expression of the VP1-2A region of HRV2 in bacteria, protein 2A was capable of acting on its own N-terminus; derived extracts specifically cleaved a 16 amino acid oligopeptide corresponding to the sequence at the cleavage site. Cleavage of the oligopeptide substrate provides a convenient in vitro assay system. Deletion experiments showed that removal of 10 amino acids from the carboxy terminus inactivated the enzyme. Site-directed mutagenesis identified an essential arginine close to the C-terminus and showed that the enzyme was sensitive to changes in the putative active site. This analysis supports the hypothesis that 2A belongs to the group of sulfhydryl proteases, although sequence comparisons indicate that the putative active site of HRV2 2A is closely related to that of the serine proteases.

A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses.

Rhinoviruses, which cause common colds, possess over 100 serotypes, 90% of which (the major group) share a single receptor. Lymphocyte function associated molecule 1 (LFA-1) mediates leukocyte adhesion to a wide variety of cell types by binding to intercellular adhesion molecule 1 (ICAM-1). We demonstrate identity between the receptor for the major group of rhinoviruses and ICAM-1. A major group rhinovirus binds specifically to purified ICAM-1 and to ICAM-1 expressed on transfected COS cells, and binding is blocked by three ICAM-1 monoclonal antibodies (MAb) that block ICAM-1-LFA-1 interaction, but not by an ICAM-1 MAb that does not block ICAM-1-LFA-1 interaction. This suggests that the ICAM-1 contact site(s) for LFA-1 and rhinoviruses is proximal or identical. In addition, ICAM-1 MAb block the cytopathic effect in HeLa cells mediated by representative major but not minor group rhinoviruses. ICAM-1 is induced by soluble mediators of inflammation, suggesting that the host immune response to rhinovirus may facilitate spread to uninfected cells.

Hybridoma-derived human suppressor factors: differential effects on mouse lymphocytes.

We have recently identified a family of suppressor factors produced by certain human T-T cell hybridomas that we developed (references 1 and 2) and by the Jurkat T cell line. These suppressor factors significantly inhibited proliferative responses to mitogens and allogeneic cells in mixed lymphocyte culture and antibody production by human peripheral blood mononuclear cells. We investigated and report here the effect of these suppressor factors on certain in vitro murine immune responses. Suppressor factors produced by certain of these hybrids, such as 153, 160, 170 and by the Jurkat T-cell line were able to inhibit: (1) proliferative responses to mitogens of mouse thymocytes and splenocytes; (2) proliferative responses of mouse splenocytes to allogeneic cells in mixed lymphocyte cultures; (3) primary in vitro antibody responses of mouse spleen lymphocytes to sheep erythrocytes; (4) primary in vitro antibody responses of mouse spleen lymphocytes to a T-cell independent antigen (TNP-Ficoll). Inhibition of murine immune responses in vitro by these suppressor factors was regular and reproducible and it was observed in a large number of experiments. In contrast, suppressor factors produced by the 169 and by the 77(38F3) hybrids did not suppress the murine immune responses. The basis for these differences are not known at the present. The ability of human suppressor factors to inhibit effectively mouse immune responses provides an additional opportunity for the characterization of the properties of these factors in vivo using mouse models of human disease.