Identification of the Fas antigen in human gingiva

The Fas antigen is a cell-surface glycoprotein that mediates apoptosis from the cell surface into the cytoplasm. Polyclonal antibody (Fas D) was raised against a synthetic polypeptide selected from the extracellular part of the human Fas antigen (amino acid residues 104-114) and was used to detect the Fas antigen in human gingiva. Biopsy specimens of human gingiva were prepared, and the paraffin sections were reacted with the Fas D antibody by an immunohistochemical method. The antibody localized to the prickle-cell layer and to granular layer keratinocytes of human gingiva. Proteins were also prepared from human gingiva and subjected to SDS-PAGE, followed by Western-blotting analysis with the Fas D antibody. The antibody interacted with a band corresponding to an estimated molecular weight of 35 kDa. The incidence of the immunoreactive 35-kDa protein was detected in the gingiva of 90% of the 20 individuals examined. The Fas antigen detected in human gingiva may be related to the physiological turnover of oral mucosa.     

Identification of the binding site in intercellular adhesion molecule 1 for its receptor, leukocyte function-associated antigen 1

Intercellular adhesion molecule 1 (ICAM-1, CD54) is a member of the Ig superfamily and is a counterreceptor for the beta 2 integrins: lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18), complement receptor 1 (MAC-1, CD11b/CD18), and p150,95 (CD11c/CD18). Binding of ICAM-1 to these receptors mediates leukocyte-adhesive functions in immune and inflammatory responses. In this report, we describe a cell-free assay using purified recombinant extracellular domains of LFA-1 and a dimeric immunoadhesin of ICAM-1. The binding of recombinant secreted LFA-1 to ICAM-1 is divalent cation dependent (Mg2+ and Mn2+ promote binding) and sensitive to inhibition by antibodies that block LFA-1-mediated cell adhesion, indicating that its conformation mimics that of LFA-1 on activated lymphocytes. We describe six novel anti-ICAM-1 monoclonal antibodies, two of which are function blocking. Thirty-five point mutants of the ICAM-1 immunoadhesin were generated and residues important for binding of monoclonal antibodies and purified LFA-1 were identified. Nineteen of these mutants bind recombinant LFA-1 equivalently to wild type. Sixteen mutants show a 66-2500-fold decrease in LFA-1 binding yet, with few exceptions, retain binding to the monoclonal antibodies. These mutants, along with modeling studies, define the LFA-1 binding site on ICAM-1 as residues E34, K39, M64, Y66, N68, and Q73, that are predicted to lie on the CDFG beta-sheet of the Ig fold. The mutant G32A also abrogates binding to LFA-1 while retaining binding to all of the antibodies, possibly indicating a direct interaction of this residue with LFA-1. These data have allowed the generation of a highly refined model of the LFA-1 binding site of ICAM-1.     

Identification of residues lining the anthrax protective antigen channel

In its activated 63 kDa form, the protective antigen (PA) component of anthrax toxin forms a heptameric prepore, which converts to a pore (channel) in endosomal membranes at low pH and mediates translocation of the toxin's enzymic moieties to the cytosol. It has been proposed that the prepore-to-pore conversion involves a conformational rearrangement of a disordered amphipathic loop (D2L2; residues 302-325), in which loops from the 7 protomers combine to form a transmembrane 14-stranded beta barrel. To test this model, we generated Cys substitutions in 24 consecutive residues of the D2L2 loop, formed channels in artificial bilayers with each mutant, and examined changes in channel conductance after adding the thiol-reactive, bilayer-impermeant reagent methanethiosulfonate ethyltrimethylammonium (MTS-ET) to the trans compartment. The rationale for these experiments is that reaction of MTS-ET with a Cys residue adds a positively charged group and therefore would likely reduce channel conductance if the residue were in the ion-conducting pathway. We found alternating reduction and absence of reduction of conductance in consecutive residues over two stretches (residues 302-311 and 316-325). This pattern is consistent with alternating polar and apolar residues of the two stretches projecting into the pore lumen and into the bilayer, respectively. Residues connecting these two stretches (residues 312-315) were responsive to MTS-ET, consistent with their being in a turn region. Single channels formed by selected mutants (H304C and N306C) showed multiple conductance step changes in response to MTS-ET, consistent with an oligomeric pore. We also found that the binding site for the channel-blocking tetraalkylammonium ions is located cis relative to the inserted D2L2 loops. These findings constitute strong evidence in favor of the model of conversion of the prepore to a 14-stranded beta barrel pore and solidify the foundation for studies to understand the mechanism of translocation by anthrax toxin.     

Identification of dipeptidyl peptidase IV as the antigen of a monoclonal anti-prostasome antibody

Two patients with previous distal splenorenal shunts (DSRSs) performed 6 years earlier underwent liver transplantation (LT). A preoperative selective mesenteric artery angiogram showed collateral veins draining mesenteric venous flow into the shunt. Intraoperative flow measurements were performed to assess the steal of portal venous flow by the shunt and determine the need for shunt occlusion. Portal vein, hepatic artery, and shunt flows were measured by ultrasound transit-time flow probes in the native liver and after graft implantation with and without temporary shunt occlusion. Hemodynamic studies showed that long-standing DSRSs are high-flow shunts that steal portal flow. After graft implantation, DSRS flows remained high. Occlusion of the shunts produced an increase in portal vein flow at an amount similar to those of splenorenal shunt. Thus, the flow measurements showed persistent steal by the shunts after graft implantation and, therefore, the DSRSs were occluded but splenectomy was not performed. We conclude that the decision to occlude a DSRS should be based on the demonstration of steal of portal flow by the shunt and reversibility once the shunt is occluded. Splenectomy is not required when the DSRS is occluded.     

Identification and characterization of the RNA chaperone activity of hepatitis delta antigen peptides

In this study, we identified an activity of the hepatitis delta antigen that both modulates the cis-cleaving activities of hepatitis delta virus (HDV) genomic RNA fragments and facilitates the trans-cleavage reactions between hammerhead ribozymes and the cognate substrates of various lengths in vitro. Hepatitis delta antigen peptides exert their effect by accelerating the unfolding and refolding of RNA molecules and by promoting strand annealing and strand dissociation. In addition, the stimulatory effect of hepatitis delta antigen peptide on hammerhead catalysis is observed whether the peptide is removed or not by phenol/chloroform extraction prior to the initiation of trans-cleavage reaction. Therefore, hepatitis delta antigen peptide acts as an RNA chaperone. The RNA chaperone domain of hepatitis delta antigen overlaps with the coiled-coil domain that is rich in lysine residues. The RNA binding domains of hepatitis delta antigen previously identified are not required for the RNA chaperone activity identified herein. The RNA chaperone activity of hepatitis delta antigen may be important for the regulation of HDV replication in vivo.     

Identification of Streptococcus mutans antigen D as the HPr component of the sugar-phosphotransferase transport system

Fosfomycin tromethamine is an orally administered fosfomycin that may be used for single-dose therapy of uncomplicated urinary tract infections. At breakpoint concentrations [< or = 128 micrograms/ml plus 25 micrograms/ml glucose-6-phosphate (G-6-P)], fosfomycin tromethamine inhibited > 90% of the 350 bacterial isolates tested. When testing Escherichia coli, Klebsiella spp., and Enterobacter spp., we note that the performance of fosfomycin disks improved when G-6-P was added to the disks. The interpretive error rates were minimized when 200-micrograms fosfomycin disks were supplemented with either 50 or 100 micrograms G-6-P. Using < or = 128 and > or = 256 micrograms/ml as the susceptible and resistant MIC breakpoints, respectively, the regression-analysis-derived disk diffusion zone diameter breakpoints for the 200-micrograms fosfomycin disk supplemented with 50 micrograms of G-6-P are as follows: susceptible, > or = 16 mm; intermediate, 13-15 mm; and resistant, < or = 12 mm.     

Identification of a graft versus host disease-associated human minor histocompatibility antigen

Minor histocompatibility antigen disparities between human leukocyte antigen (HLA)-matched bone marrow donors and recipients are a major risk factor for graft versus host disease (GVHD). An HLA-A2.1-restricted cytotoxic T cell clone that recognized the minor histocompatibility antigen HA-2 was previously isolated from a patient with severe GVHD after HLA-identical bone marrow transplantation. The HLA-A2.1-bound peptide representing HA-2 has now been identified. This peptide appears to originate from a member of the non-filament-forming class I myosin family. Because HA-2 has a phenotype frequency of 95 percent in the HLA-A2.1-positive population, it is a candidate for immunotherapeutic intervention in bone marrow transplantation.     

Identification of a Coccidioides immitis antigen 2 domain that expresses B-cell-reactive epitopes

Antigen 2 (Ag2), a major immunoreactive component of Coccidioides immitis mycelium- and spherule-phase cell walls, was recently cloned in our laboratory and was shown to elicit T-cell responses in Coccidioides-immune mice. In this investigation, we evaluated recombinant Ag2 (rAg2) and PCR-generated Ag2 truncations for expression of B-cell-reactive epitopes in enzyme-linked immunosorbent and immunoblot assays with sera from patients with active coccidioidomycosis, a hyperimmune goat anti-Ag2 serum, and a murine anti-Ag2 monoclonal antibody that recognizes a conformational epitope. The results established that rAg2 expresses both linear and conformational B-cell-reactive epitopes which are localized to a domain comprised of amino acids 19 through 96 (designated A19-96). Truncations designed to identify epitopes within the A19-96 domain yielded fragments that either were nonreactive (A62-194, A19-61, and A49-79) or showed reduced reactivity (A19-79). Hence, A19-96 was the shortest domain expressing epitopes recognized by the panel of antibodies. The prevalence of antibodies to the A19-96 domain was evaluated in enzyme-linked immunosorbent assays of sera from 28 coccidioidomycosis patients. Antibody reactivity was detected in 79% of the patients' sera, and the level of antibody reactivity was directly correlated with disease severity. Whereas patients with pulmonary disease showed a mean response (A405) of 0.16 +/- 0.04, patients with disseminated coccidioidomycosis showed a mean response of 0.69 +/- 0.17 (P < 0.05). No reactivity was detected with sera from histoplasmosis or blastomycosis patients. The production of a recombinant peptide that expresses C. immitis-specific Ag2 epitopes provides a useful reagent for examining the role of anti-Ag2 antibodies in coccidioidomycosis.     

Activation of the Rap1 GTPase by the B cell antigen receptor

The B cell antigen receptor (BCR) activates Ras, a GTPase that promotes cell proliferation by activating the Raf-1/MEK/ERK signaling module and other signaling enzymes. In its active GTP-bound form, the Rap1 GTPase may act as a negative regulator of Ras-mediated signaling by sequestering Ras effectors (e.g., Raf-1) and preventing their activation. In this report, we show that BCR engagement activates Rap1 and that this is dependent on production of diacylglycerol (DAG) by phospholipase C-gamma. Activation of Rap1 by the BCR was greatly reduced in phospholipase C-gamma-deficient B cells, whereas both a synthetic DAG and phorbol dibutyrate could activate Rap1 in B cells. We had previously shown that C3G, an activator of Rap1, associates with the Crk adaptor proteins in B cells and that BCR engagement causes Crk to bind to the Cas and Cbl docking proteins. However, the DAG-dependent pathway by which the BCR activates Rap1 apparently does not involve Crk signaling complexes since phorbol dibutyrate could activate Rap1 without inducing the formation of these complexes. Thus, the BCR activates Rap1 via a novel DAG-dependent pathway.     

Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen

In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp. The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco. bovis (bacille Calmette-Guérin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts. In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay. Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco. leprae hsp70-reactive individuals responded to C-344. Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344. The smaller C-142 fragment which includes the terminal 70 residues unique to Myco. leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees. In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared. Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes. The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides. Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes. Although both were located in regions of the molecule shared with Myco. tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.     

Identification of 45 kD antigen in immune complexes of patients of allergic bronchopulmonary aspergillosis

The hippocampus is a major target of alpha-bungarotoxin (alpha-BTX) binding. This ligand binds to the alpha 7 nicotinic, cholinergic receptor, which has been implicated in hippocampal habituation to repetitive auditory stimulation, a phenomenon thought to involve inhibitory neurons. This study examined whether alpha-BTX binds to neurons containing nitric oxide synthase (NOS), a marker of one subgroup of inhibitory hippocampal neurons. Rat hippocampal sections were processed for NOS immunohistochemistry, photographed and then processed for [125I]alpha-BTX autoradiography. Comparison between the distribution of neurons immunoreactive for NOS and those positive for alpha-BTX binding in the same regions of the hippocampal formation revealed a variable degree of colocalization of NOS and alpha-BTX. Of the cells labeled with alpha-BTX, 2% in the dentate gyrus and 40% in the hippocampus proper were also immunoreactive for NOS. These NOS/alpha-BTX neurons were most prevalent in CA1 stratum oriens. The results suggest a possible role for NOS-containing neurons in alpha 7-mediated inhibition to repetitive auditory stimulation in rat hippocampus.     

Neutrophil-specific antigen NB1 inhibits neutrophil-endothelial cell interactions

Neutrophil-specific antigen NB1 is located on a 58 to 64 kd glycosyl phosphatidylinositol-linked plasma membrane glycoprotein. NB1 antigen can be detected on neutrophils from 97% of healthy volunteers, and NB1 antigen is expressed on subpopulations of neutrophils. Neutrophil subpopulations with varying functions have been described, and we hypothesize that NB1 antigen may play an important role in neutrophil function. We compared the function of NB1-positive and NB1-negative neutrophils obtained from several persons. There were no differences in the adhesion of NB1-positive and NB1-negative neutrophils incubated in C5a, N-formyl-Met-Leu-Phe (FMLP), phorbol myristate acetate (PMA), or buffer to type IV collagen, fibronectin, laminin, or polystyrene. However, the adherence to human umbilical vein endothelial cells (HUVEC) monolayers of unstimulated NB1-positive neutrophils was less than to NB1-negative neutrophils (20.0% +/- 4.2% vs 31.7% +/- 5.8%; p < 0.01). When neutrophils were stimulated with C5a, PMA, or FMLP, no differences were found in the adhesion of NB1-positive and NB1-negative cells to the same surfaces. When NB1-positive neutrophils were incubated with rabbit polyclonal anti-NB1 Fab fragments, their adherence to HUVEC was increased (32.9% +/- 10.1% vs 18.3% +/- 5.0%; p < 0.05). Fab fragments prepared from normal rabbit serum had no effect on neutrophil adherence to HUVEC. The chemotaxis of NB1-positive neutrophils to FMLP through nitrocellulose was significantly greater than that of NB1-negative neutrophils (p = 0.03), but there was no difference in chemotaxis to FMLP through polycarbonate membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of epitopes on the 120-kilodalton surface protein antigen of Rickettsia prowazekii with synthetic peptides

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae are highly immunogenic and have been shown to be responsible for the species-specific serological reactions of the typhus group rickettsiae. To study the immunochemistry of these proteins, overlapping decapeptides encompassing the whole protein were synthesized on derivatized polyethylene pins. A modified enzyme-linked immunosorbent assay was used to identify epitopes recognized by rabbit hyperimmune antisera to Rickettsia prowazekii SPA. Eight distinct epitopes were mapped by this method in three regions. Four of the epitopes, which were located in the carboxyterminus of mature processed SPA, were strongly competitively inhibited by native folded SPA but not by intact rickettsiae, suggesting that they were on the SPA surface but not exposed on the rickettsial surface. Three of these epitopes were present on both R. prowazekii and Rickettsia typhi SPAs. The immunoreactivities of five epitopes were further characterized by synthesizing modified peptides. Glycine substitution experiments determined the critical residues in the epitopes. The dependence of binding of the peptide epitopes to the polyclonal antisera was mapped to single residues. The limited number and weak reactivity of linear peptide epitopes observed with human and rabbit sera, possibly due to a lack of the methylated amino acids which are present in rickettsia-derived SPA, suggest that the present approach will not provide useful synthetic antigens for diagnosis of typhus infections.     

AgSK1, a novel carcinoma associated antigen

An IgM human monoclonal antibody (HuMAb) SK1 was generated from mesenteric nodal lymphocytes of a colon cancer patient that were fused with a human B-lymphoblastoid cell line SHFP-1. The reactivities of HuMAb SK1 to various human cell lines were screened by cell enzyme linked immunosorbent assay and immunocytochemical staining. The HuMAb SK1 reacted strongly with all 11 human carcinoma cell lines that were tested and had no detectable binding with noncarcinoma cell lines of the following origins: fibroblast; fetal lung; melanoma; soft tissue sarcoma; neuroblastoma; and glioblastoma. Carcinoma preferred reactivity of HuMAb SK1 was further confirmed by immunoperoxidase staining of a large number of frozen tissues, both malignant and benign. The antigen SK1 (AgSK1) in human carcinoma detected by immunoperoxidase staining was also identified biochemically as a sialoglycoprotein that migrated at M(r) 42,000 with an isoelectric point (pI) of approximately 5.9. A preferential staining by HuMAb SK1 was seen among colorectal, gastric, pancreatic, and lung cancers. Competitive inhibition study in solid-phase immunoassay suggested that the HuMAb SK1 did not cross-react with other antibodies specific for CEA, CA 19-9, and TAG 72. The AgSK1 appears to be a novel carcinoma associated antigen which may be a useful tumor marker in cancer diagnosis and treatment.     

Identification of a new Pyk2 isoform implicated in chemokine and antigen receptor signaling

Pyk2 is a protein tyrosine kinase that links G-protein-coupled receptors, inflammatory cytokines, and extracellular stimuli that elevate intracellular calcium concentration with activation of the mitogen-activated protein kinase pathways and regulation of ion channel functions. Here we describe the identification, cloning, and characterization of a new isoform of Pyk2 (Pyk2-H) that is generated by alternative RNA splicing. Pyk2-H is mainly expressed in hematopoietic cells including T-cells, B-cells, and natural killer cells. Engagement of T-cell or B-cell antigen receptors leads to rapid tyrosine phosphorylation of Pyk2-H. Pyk2-H is also activated in response to the chemokines RANTES and macrophage inflammatory protein-1beta in T cells. In addition, we show that glutathione S-transferase fusion proteins containing the carboxyl termini of Pyk2 and Pyk2-H bind to a different set of tyrosine-phosphorylated proteins in thymus lysates. Specific expression of Pyk2-H and its activation by antigens or chemokines in hematopoietic cells may contribute toward the generation of cell type-specific signals involved in host immune responses.     

Identification of HLA-A2-restricted epitopes of the tumor-associated antigen MUC2 recognized by human cytotoxic T cells

BACKGROUND: Dopexamine is a specific dopaminergic and beta2-adrenergic agonist. Using newborn piglets, we have previously shown that (1) dopexamine increases cardiac output and mesenteric blood flow; (2) indomethacin reduces mesenteric blood flow. METHODS: Ultrasonic blood flow probes were placed around the ascending aorta, cranial mesenteric artery, and a renal artery of 0 to 2-day-old and 2-week-old piglets. Animals of each age were grouped (5 to 8 animals per group) and subjected to one of three experimental protocols: (1) 0.4 mg/kg indomethacin infusion, (2) 10 microg/kg/min dopexamine infusion begun 10 minutes before indomethacin, or (3) no treatment. RESULTS: Control animals demonstrated no significant alterations in mesenteric blood flow. Compared with baseline, indomethacin produced significant (P< .05, analysis of variance) declines in cranial mesenteric artery blood flow in 0 to 2-day old (37.2+/-5.7 mL/min v 17.9+/-3.7 mL/min at 90 min), and 2-week-old (80.2+/-12.5 mL/min v 29.7+/-5.7 mL/min at 90 minutes) piglets. In both animal groups treated with dopexamine before indomethacin, the decreases in cranial mesenteric artery blood flow were eliminated (38.4+/-7.6 mL/min at baseline v 36.5+/-6.8 mL/min at 90 minutes in 0 to 2 day olds; 79.9+/-10.0 mL/min at baseline v 77.5+/-14.7 mL/min in 2 week olds). Indomethacin-induced declines in renal blood flow were similarly abrogated by dopexamine. CONCLUSION: Dopexamine may prove of clinical benefit when a neonate is considered a candidate for indomethacin therapy.