Identification of C1q as the heat-labile serum cofactor required for immune complexes to stimulate endothelial expression of the adhesion molecules E-selectin and intercellular and vascular cell adhesion molecules 1.

To examine the role of complement components as regulators of the expression of endothelial adhesive molecules in response to immune complexes (ICs), we determined whether ICs stimulate both endothelial adhesiveness for leukocytes and expression of E-selectin and intercellular and vascular cell adhesion molecules 1 (ICAM-1 and VCAM-1). We found that ICs [bovine serum albumin (BSA)-anti-BSA] stimulated endothelial cell adhesiveness for added leukocytes in the presence of complement-sufficient normal human serum (NHS) but not in the presence of heat-inactivated serum (HIS) or in tissue culture medium alone. Depletion of complement component C3 or C8 from serum did not prevent enhanced endothelial adhesiveness stimulated by ICs. In contrast, depletion of complement component C1q markedly inhibited IC-stimulated endothelial adhesiveness for leukocytes. When the heat-labile complement component C1q was added to HIS, the capacity of ICs to stimulate endothelial adhesiveness for leukocytes was completely restored. Further evidence for the possible role of C1q in mediating the effect of ICs on endothelial cells was the discovery of the presence of the 100- to 126-kDa C1q-binding protein on the surface of endothelial cells (by cytofluorography) and of message for the 33-kDa C1q receptor in resting endothelial cells (by reverse transcription-PCR). Inhibition of protein synthesis by cycloheximide blocked endothelial adhesiveness for leukocytes stimulated by either interleukin 1 or ICs in the presence of NHS. After stimulation with ICs in the presence of NHS, endothelial cells expressed increased numbers of adhesion molecules (E-selectin, ICAM-1, and VCAM-1). Endothelial expression of adhesion molecules mediated, at least in part, endothelial adhesiveness for leukocytes, since leukocyte adhesion was blocked by monoclonal antibodies directed against E-selectin. These studies show that ICs stimulate endothelial cells to express adhesive proteins for leukocytes in the presence of a heat-labile serum factor. That factor appears to be C1q.

GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia

High-affinity (Kd = 1 × 10−9 M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC50 = 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.

Identification and characterization of a human protein kinase related to budding yeast Cdc7p

The Cdc7p protein kinase is essential for the G1/S transition and initiation of DNA replication during the cell division cycle in Saccharomyces cerevisiae. Cdc7p appears to be an evolutionarily conserved protein, since a homolog Hsk1 has been isolated from Schizosaccharomyces pombe. Here, we report the isolation of a human cDNA, HsCdc7, whose product is closely related in sequence to Cdc7p and Hsk1. The HsCdc7 cDNA encodes a protein of 574 amino acids with predicted size of 64 kDa. HsCdc7 contains the conserved subdomains common to all protein-serine/threonine kinases and three “kinase inserts” that are characteristic of Cdc7p and Hsk1. Immune complexes of HsCdc7 from cell lysates were able to phosphorylate histone H1 in vitro. Indirect immunofluorescence staining demonstrated that HsCdc7 protein was predominantly localized in the nucleus. Although the expression levels of HsCdc7 appeared to be constant throughout the cell cycle, the protein kinase activity of HsCdc7 increased during S phase of the cell cycle at approximately the same time as that of Cdk2. These results, together with the functions of Cdc7p in yeast, suggest that HsCdc7 may phosphorylate critical substrate(s) that regulate the G1/S phase transition and/or DNA replication in mammalian cells.

Akt phosphorylates and regulates the orphan nuclear receptor Nur77

The immediate early gene NUR77 (also called NGFI-B) is required for T cell antigen receptor-mediated cell death and is induced to very high levels in immature thymocytes and T cell hybridomas undergoing apoptosis. The Akt (PKB) kinase is a key player in transduction of anti-apoptotic and proliferative signals in T cells. Because Nur77 has a putative Akt phosphorylation site at Ser-350, and phosphorylation of this residue is critical for the transactivation activity of Nur77, we investigated whether Akt regulates Nur77. Coimmunoprecipitation experiments showed the detection of Nur77 in Akt immune complexes, suggesting that Nur77 and Akt physically interact. We further show that Akt specifically phosphorylates Ser-350 of the Nur77 protein within its DNA-binding domain in vitro and in vivo in 293 and NIH 3T3 cells. Because phosphorylation of Ser-350 of Nur77 is critical for its function as a transcription factor, we examined the effect of Akt on this function. By using luciferase assay experiments, we showed that phosphorylation of Nur77 by Akt decreased the transcriptional activity of Nur77 by 50–85%. Thus, we show that Akt interacts with Nur77 and inactivates Nur77 by phosphorylation at Ser-350 in a phosphatidylinositol 3-kinase-dependent manner, connecting the phosphatidylinositol 3-kinase-dependent Akt pathway and a nuclear receptor pathway.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA–liposome complexes

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA–liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Superactivation of an immune response triggered by oligomerized T cell epitopes

The peptides bound to class II major histocompatibility complex (MHC) molecules extend out both ends of the peptide binding groove. This structural feature provided the opportunity to design multivalent polypeptide chains that cross-link class II MHC molecules through multiple, repetitive MHC binding sites. By using recombinant techniques, polypeptide oligomers were constructed that consist of up to 32 copies of an HLA-DR1-restricted T cell epitope. The epitope HA306–318, derived from influenza virus hemagglutinin, was connected by 12- to 36-aa long spacer sequences. These oligomers were found to cross-link soluble HLA-DR1 molecules efficiently and, upon binding to the MHC molecules of a monocyte line, to trigger signal transduction indicated by the enhanced expression of some cell surface molecules. A particularly strong effect was evident in the T cell response. A hemagglutinin-specific T cell clone recognized these antigens at concentrations up to three to four orders of magnitude lower than that of the peptide or the hemagglutinin protein. Both signal transduction in the monocyte and the proliferative response of the T cell were affected greatly by the length of the oligomer (i.e., the number of repetitive units) and the distance of the epitopes within the oligomer (spacing). Thus, the formation of defined clusters of T cell receptor/MHC/peptide antigen complexes appears to be crucial for triggering the immune response and can be used to enhance the antigenicity of a peptide antigen by oligomerizing the epitope.

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.