The plant lectin wheat germ agglutinin inhibits the binding of pemphigus foliaceus autoantibodies to desmoglein 1 in a majority of patients and prevents pathomechanisms of pemphigus foliaceus in vitro and in vivo

Pemphigus foliaceus (PF) is a life-threatening autoimmune blistering skin disease caused by pathogenic IgG autoantibodies against desmoglein 1 (dg1), a desmosomal cadherin-type adhesion glycoprotein. Using lectins and glycosidases, we have shown that dg1 displays an N-glycosylation pattern of the complex triantennary type. We have found that lectins and glycosidases interfere with N-bound sugar residues on the amino-terminal ectodomain of dg1 and completely abolish, in vitro, the antigenicity of dg1 in most of the patients' sera. Moreover, in an ex vivo model using punch biopsies from normal human skin, we demonstrate that preincubation of the epidermis in wheat germ agglutinin (WGA) prevents PF autoantibody binding, acantholysis, and subcorneal blistering. In addition, we show that topical treatment with WGA inhibits PF autoantibody binding to keratinocytes in both newborn BALB/c mice and in organotypic human epidermis grafted onto the back of SCID mice. The epidermis of these pretreated animals displays a regular morphology, whereas control animals develop the immunopathologic phenotype of PF. These findings suggest that WGA may interfere with autoantibody binding to dg1, preventing experimental PF without affecting the adhesive function of dg1. Our observations may provide a new approach to the therapy of PF.     

Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3

Pemphigus vulgaris (PV) is a life-threatening autoimmune blistering disease that is caused by IgG autoantibodies against the cadherin-type adhesion molecule desmoglein (Dsg)3. Previously, we have generated an active mouse model for PV by adoptive transfer of Dsg3(-/-) splenocytes. In this study, we isolated eight AK series, anti-Dsg3 IgG mAbs from the PV mouse model, and examined their pathogenic activities in induction of blister formation. Intraperitoneal inoculation of the AK23 hybridoma, but not the other AK hybridomas, induced the virtually identical phenotype to that of PV model mice or Dsg3(-/-) mice with typical histology of PV. Epitope mapping with domain-swapped and point-mutated Dsg1/Dsg3 molecules revealed that AK23 recognized a calcium-dependent conformational epitope on Dsg3, which consisted of the V3, K7, P8, and D59 Dsg3-specific residues that formed the adhesive interface between juxtaposed Dsg, as predicted by the crystal structure. The epitopes of the mAbs that failed to show apparent pathogenic activity were mapped in the middle to carboxyl-terminal extracellular region of Dsg3, where no direct intermolecular interaction was predicted. These findings demonstrate the pathogenic heterogeneity among anti-Dsg3 IgG Abs due to their epitopes, and suggest the direct inhibition of adhesive interaction of Dsg as an initial molecular event of blister formation in pemphigus.     

A monoclonal antibody to a synthetic fragment of rabies virus glycoprotein binds ligands of the nicotinic cholinergic receptor

Rabies virus glycoprotein and snake venom curaremimetic neurotoxins share a region of high homology (30-45 for neurotoxins and 190-203 for the glycoprotein) in the regions that are believed to be responsible for binding the nicotinic acetylcholine receptor. Monoclonal antibodies raised to the 190-203 synthetic fragment of rabies virus glycoprotein were immobilized on a high performance affinity chromatography column and were able to bind neurotoxins. Toxins were displaced from the affinity column by elution at acidic pH and by affinity competition with acetylcholine at neutral pH. Furthermore, the affinity column proved to be useful for the purification of cholinergic ligands. Overall, these results indicate that the paratope of our monoclonal antibodies could behave as an 'internal image' of the nicotinic cholinergic receptor acetylcholine binding site.     

Serum from pemphigus vulgaris reduces desmoglein 3 half-life and perturbs its de novo assembly to desmosomal sites in cultured keratinocytes

Defects of cell-cell adhesion underlie disruption of epithelial integrity observed in patients with pemphigus vulgaris (PV), an autoimmune disease characterized by severe mucosal erosions and skin blisters. Pathogenic PV autoantibodies found in patients' sera target desmoglein 3 (Dsg3), a major component of the desmosome, but how does this phenomenon affect Dsg-dependent adhesion and lead to acantholysis still remains controversial. Here, we show that PV serum determines a reduction of Dsg3 half-life in HaCaT keratinocytes, although the total amount of Dsg3 remains unchanged. Immunofluorescence studies suggest that PV IgG exert their effect prevalently by binding non-desmosomal Dsg3 without causing its massive internalization. Furthermore, PV IgG targeting desmosome-assembled Dsg3 do not induce depletion of Dsg3 from the adhesion sites. Conversely, incorporation of PV IgG-Dsg3 complexes into new forming desmosomes appears perturbed. With our study, the basic biochemical changes of Dsg3 in an in vitro model of PV have been defined.     

A monoclonal antibody to triplex DNA binds to eucaryotic chromosomes.

A monoclonal antibody (Jel 318) was produced by immunizing mice with poly[d(TmC)].poly[d(GA)].poly[d(mCT) which forms a stable triplex at neutral pH. Jel 318 did not bind to calf thymus DNA or other non pyrimidine.purine DNAs such as poly[d(TG)].poly[d(CA)]. In addition the antibody did not recognize pyrimidine.purine DNAs containing mA (e.g. poly[d(TC)].poly[d(GmA)]) which cannot form a triplex since the methyl group blocks Hoogsteen base-pairing. The binding of Jel 318 to chromosomes was assessed by immunofluorescent microscopy of mouse myeloma cells which had been fixed in methanol/acetic acid. An antibody specific for duplex DNA (Jel 239) served as a control. The fluorescence due to Jel 318 was much weaker than that of Jel 239 but binding to metaphase chromosomes and interphase nuclei was observed. The staining by Jel 318 was unaffected by addition of E. coli DNA but it was obliterated in the presence of triplex. Since an acid pH favours triplex formation, nuclei were also prepared from mouse melanoma cells by fixation in cold acetone. Again Jel 318 showed weak but consistent staining of the nuclei. Therefore it seems likely that triplexes are an inherent feature of the structure of eucaryotic DNA.     

A monoclonal anti-idiotypic antibody against a human monoclonal IgM with specificity for myelin-associated glycoprotein

A human monoclonal IgM lambda antibody, directed against MAG, obtained from a patient with polyneuropathy associated with a gammopathy, was used as an immunogen to generate mouse monoclonal anti-idiotype antibodies. One hybridoma antibody, designated A8F2, reacts uniquely with the M-IgM of the patient, shows high affinity binding to the patient's M-IgM, and dose-dependently inhibits binding of the patient's M-IgM to its specific antigen MAG. Thus, A8F2 is a monoclonal anti-idiotype antibody that recognizes a region of the MAG binding site of the patient's IgM. Use of this anti-idiotype antibody in a competition RIA revealed the presence of naturally occurring anti-idiotype in the patient's serum. Because anti-idiotype antibodies may be part of a mechanism for down-regulation of antibody production, the use of A8F2 to induce a specific immunosuppression should be considered.     

Identification of desmoglein, a constitutive desmosomal glycoprotein, as a member of the cadherin family of cell adhesion molecules.

Monoclonal antibodies to the constitutive desmosomal glycoprotein desmoglein were characterized whose epitopes are located intracellularly, i.e., in the cytoplasmic portion of this molecule, and contribute to the structure of the desmosomal plaque. Using one of these antibodies (DG3.10), a peptide was isolated from a proteolytic digest of desmoglein purified from isolated bovine muzzle demosomes, and its amino acid sequence was determined. In comparisons of this sequence with the amino acid sequence of desmoglein as deduced from the sequence of cDNA clones from the same tissue, encompassing most of approximately 7.6 kb mRNA and the complete coding region of 959 residues (calculated molecular weight approximately 102,400), the DG3.10 epitope was identified in a region starting 163 amino acids before the carboxy terminus in the first of four consecutive repeats of a homologous element of 29 +/- 1 amino acids. This topological information, together with the identification of a single hydrophobic region of sufficient length to provide a transmembrane segment and of several extended regions showing high sequence homology to various cadherins, has allowed the construction of a model of the molecular organization of desmoglein. We conclude that desmoglein is a member of the cadherin family of cell adhesion glycoproteins which is characterized by an unusually long cytoplasmic domain which exceeds those of the cadherins by more than 275 amino acids, contains special repetitive elements and spans the desmosomal plaque at least once.     

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. I. Biochemical identification of the polypeptide

Two murine monoclonal antibodies (DG 3.4 and DG 3.10) raised against a major glycoprotein ("band 3 component") from desmosomes of bovine muzzle epidermis were used in immunoblot experiments following SDS-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis to identify this or immunologically related proteins in other bovine tissues and cultured cell lines. In all desmosome-bearing cells, i.e. cells also expressing desmoplakins, including representative of stratified, transitional and simple epithelia as well as myocardium, only a single distinct polypeptide of identical Mr value (165,000) and electrical charge was detected. These findings, together with the immunolocalization results reported in the companion paper indicate that this glycoprotein (desmoglein) is a general constituent protein of desmosomes, providing a case of an integral membrane protein co-expressed with non-membranous desmosomal proteins such as the plaque component, desmoplakin I. Our results further suggest that, contrary to previous suggestions, desmoglein is very similar, if not identical in different cells of the same species and does not display significant cell type diversity.     

A monoclonal antibody to human insulin receptor

A murine hybridoma secreting antibody against human insulin receptor was produced by fusing FO myeloma cells with spleen and lymph node cells from a mouse that had been immunized with insulin receptor purified from human placenta. The secreted antibody was an IgG₁ (κ), designated αIR-1. Like the previously described rabbit polyclonal antibody, αIR-1 did not inhibit insulin binding. It specifically immunoprecipitated ¹²⁵I-insulin-receptor complexes as well as unoccupied receptor previously labeled directly with lactoperoxidase. Thus, αIR-1 interacts with the receptor at a site distinct from the insulin binding site. Unlike previously described anti-insulin receptor antibodies, αIR-1 exhibits strong tissue and species specificity.     

The human cytomegalovirus US8 glycoprotein binds to major histocompatibility complex class I products

Human cytomegalovirus US8 is a type I membrane protein that partially colocalizes with cellular endosomal and lysosomal proteins. Although US8 does not have discernible effects on the processing and cell surface distribution of major histocompatibility complex (MHC) class I products, we have demonstrated that US8 binds to MHC class I heavy chains in the endoplasmic reticulum.     

The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide

Summary Human spasmolytic polypeptide (hSP) is a member of the growing family of trefoil peptides which are expressed in discrete regions of the body, most notably the gastrointestinal tract. Much of the research into the localization of the spasmolytic polypeptide has relied on hybridization in situ to detect its mRNA, due to the absence of a suitable antibody. The aim of the present study was to develop and characterize a monoclonal antibody against the human spasmolytic polypeptide, using a combination of immunohistochemistry and hybridization in situ.After immunoblotting, the antibody detected a 14 kDa protein in gastrointestinal tissue extracts from the stomach and small intestine only. Using immunohistochemistry, human spasmolytic polypeptide showed a distinctive staining pattern in the duodenum which co-localized with its mRNA. The co-localization of the immunoreactive peptide with its mRNA provides good evidence that the antibody truly recognized human spasmolytic polypeptide.     

A new monoclonal antibody, 4-1a,that binds to the amino terminus of human lipoprotein lipase

Lipoprotein lipase (LPL) has been highly conserved through vertebrate evolution, making it challenging to generate useful antibodies. Some polyclonal antibodies against LPL have turned out to be nonspecific, and the available monoclonal antibodies (Mabs) against LPL, all of which bind to LPL's carboxyl terminus, have drawbacks for some purposes. We report a new LPL-specific monoclonal antibody, Mab 4-1a, which binds to the amino terminus of LPL (residues 5–25). Mab 4-1a binds human and bovine LPL avidly; it does not inhibit LPL catalytic activity nor does it interfere with the binding of LPL to heparin. Mab 4-1a does not bind to human hepatic lipase. Mab 4-1a binds to GPIHBP1-bound LPL and does not interfere with the ability of the LPL–GPIHBP1 complex to bind triglyceride-rich lipoproteins. Mab 4-1a will be a useful reagent for both biochemists and clinical laboratories.     

A sensitive radioimmunoassay of alpha human atrial natriuretic polypeptide using monoclonal antibody recognizing human form ring structure

A monoclonal antibody (C351) against alpha human atrial natriuretic polypeptide (alpha hANP) recognizing human form ring structure was established and applied to a radioimmunoassay of plasma alpha hANP. The minimum detectable amount in terms of 10% radioligand displacement relative to zero dose were 0.28 fmol/tube, corresponding to 0.7 fmol/ml in plasma after extraction using Sep-Pak C18 cartridges. When the mean plasma levels at recumbent position in fasted morning were compared in 10 young (less than 30 years) and 10 elderly (greater than or equal to 50 years) healthy subjects taking normal sodium diet, it was slightly higher in the latter (3.2 +/- 0.4 vs 4.7 +/- 0.5 fmol/ml, mean +/- SE, p less than 0.05). After i.v. infusion of hypertonic saline (2.5% NaCl) at a rate of 0.24 ml/kg/min for 20 min in 6 normal subjects (26 to 35 years), it was increased from 4.1 +/- 0.4 to 5.9 +/- 0.7 fmol/ml (p less than 0.01). In 6 patients with essential hypertension (34 to 57 years), it was elevated with high salt intake, i.e. 3.3 +/- 0.3, 3.9 +/- 1.03 and 7.6 +/- 1.5 fmol/ml under 34, 170 and 340 mEq NaCl/day for 7 days, respectively. From these results, the radioimmunoassay of plasma IR-alpha hANP using MAb C351 seems to be quite suitable to detect rather small changes at low plasma concentrations and to investigate a physiological importance of alpha hANP in man.     

A monoclonal antibody identifies a glycoprotein complex involved in cell-substratum adhesion

The monoclonal antibody CSAT has been reported to perturb the adhesion of chick embryo cells to their substratum (Neff et al. [19]). Evidence is presented here that the antigen recognized by this monoclonal antibody is comprised of three membrane glycoproteins. The antigen is released from cells with non-ionic detergent and purified by monoclonal antibody affinity chromatography. When analysed by SDS-PAGE under non-reducing conditions, the antigen resolves into three components of apparent molecular weights 160 000 (band 1), 135000 (band 2), and 110 000 (band 3). Following reduction of each component, bands 1 and 2 migrate at slightly lower apparent molecular weights, while band 3 migrates at a higher apparent molecular weight, suggesting that band 3 has an internal disulfide bond. All three bands differ from one another as determined by peptide mapping and by immunologic cross-reactivity. It is postulated that the three glycoproteins function as a complex that plays a central role in cell-substratum adhesion.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.     

A monoclonal antibody specific for the 200 K polypeptide of the neurofilament triplet

A mouse monoclonal antibody, designated NF1, was obtained from a cloned hybridoma isolated from a fusion of mouse myeloma Sp2 cells with spleen cells from a BALB/c mouse immunized with a crude neurofilament preparation from porcine spinal cord. NF1 is an IgG1 and recognizes, in immune blotting procedures, only the 200 K neurofilament triplet component. Its neurofilament-specific nature is further revealed by immunofluorescence microscopy studies on frozen tissue sections and various cultured cells. Immunoelectron microscopy studies on cytoskeletons of cultured neurones emphasize the discontinuous display along each neurofilament previously observed with polyclonal antibodies specific for the 200 K component after appropriate but rather cumbersome cross-absorption steps. Use of NF1 on various neuronal cells strongly supports the previous proposal of the existence of certain subpopulations of neurofilament-free neurones and the observation that certain neuronal arrangements, (e.g., those in dendrites of pyramidal cells of the hippocampus), although rich in neurofilaments, probably lack the normal 200 K triplet component. Since NF1 shows a broad cross-species reactivity and is able to react on formaldehyde-fixed tissue, it should be a useful reagent to study differential neurofilament expression and organization in embryonic, adult and pathological tissues.     

A hybridoma producing human monoclonal antibody specific for glycoprotein 120 kDa of human immunodeficiency virus (HIV-1

A stable hybridoma producing anti-HIV human monoclonal antibody (HMCA) was generated by fusing CD3-depleted human splenic lymphocytes from an HIV sero-positive donor with the mouse myeloma cell line P3x63AgU1. The resultant hybridoma has been secreting IgG1, lambda chain for over nine months at a rate of 2.5 micrograms/10(6)cells/day. The HMCA shows specific reactivity in ELISA using HIV-infected cell lysates. Immunofluorescence tests have indicated that this HMCA binds specifically to the surface of H9 and C3 HIV/HTLVIIIb infected cells, HIV/N1T infected CEM cells and to MoT cells infected with an HIV clinical isolate. Western blotting revealed recognition of glycoproteins 120 and 160 kDa of HIV by the HMCA. Although this HMCA demonstrated no neutralizing activity, the production of an anti-HIV HMCA specific for glycoprotein 120 kDa indicates the possibility that a neutralizing HMCA can be developed as further fusions with lymph nodes and spleens from HIV positive donors are performed.     

Characterization and epitope mapping of a human monoclonal antibody reactive with the envelope glycoprotein of human immunodeficiency virus

A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.