Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2

Mutations are introduced into rearranged Ig variable genes at a frequency of 10−2 mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2−/− human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.

The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK

It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995) Science 270, 1988–1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.

Analysis of the role of the Spitzenkörper in fungal morphogenesis by computer simulation of apical branching in Aspergillus niger

High-resolution video microscopy, image analysis, and computer simulation were used to study the role of the Spitzenkörper (Spk) in apical branching of ramosa-1, a temperature-sensitive mutant of Aspergillus niger. A shift to the restrictive temperature led to a cytoplasmic contraction that destabilized the Spk, causing its disappearance. After a short transition period, new Spk appeared where the two incipient apical branches emerged. Changes in cell shape, growth rate, and Spk position were recorded and transferred to the fungus simulator program to test the hypothesis that the Spk functions as a vesicle supply center (VSC). The simulation faithfully duplicated the elongation of the main hypha and the two apical branches. Elongating hyphae exhibited the growth pattern described by the hyphoid equation. During the transition phase, when no Spk was visible, the growth pattern was nonhyphoid, with consecutive periods of isometric and asymmetric expansion; the apex became enlarged and blunt before the apical branches emerged. Video microscopy images suggested that the branch Spk were formed anew by gradual condensation of vesicle clouds. Simulation exercises where the VSC was split into two new VSCs failed to produce realistic shapes, thus supporting the notion that the branch Spk did not originate by division of the original Spk. The best computer simulation of apical branching morphogenesis included simulations of the ontogeny of branch Spk via condensation of vesicle clouds. This study supports the hypothesis that the Spk plays a major role in hyphal morphogenesis by operating as a VSC—i.e., by regulating the traffic of wall-building vesicles in the manner predicted by the hyphoid model.

Characterization of anti-anti-idiotypic antibodies that bind antigen and an anti-idiotype

Two mouse monoclonal anti-anti-idiotopic antibodies (anti-anti-Id, Ab3), AF14 and AF52, were prepared by immunizing BALB/c mice with rabbit polyclonal anti-idiotypic antibodies (anti-Id, Ab2) raised against antibody D1.3 (Ab1) specific for the antigen hen egg lysozyme. AF14 and AF52 react with an “internal image” monoclonal mouse anti-Id antibody E5.2 (Ab2), previously raised against D1.3, with affinity constants (1.0 × 109 M−1 and 2.4 × 107 M−1, respectively) usually observed in secondary responses against protein antigens. They also react with the antigen but with lower affinity (1.8 × 106 M−1 and 3.8 × 106 M−1). This pattern of affinities for the anti-Id and for the antigen also was displayed by the sera of the immunized mice. The amino acid sequences of AF14 and AF52 are very close to that of D1.3. In particular, the amino acid side chains that contribute to contacts with both antigen and anti-Id are largely conserved in AF14 and AF52 compared with D1.3. Therapeutic immunizations against different pathogenic antigens using anti-Id antibodies have been proposed. Our experiments show that a response to an anti-Id immunogen elicits anti-anti-Id antibodies that are optimized for binding the anti-Id antibodies rather than the antigen.

Antibodies to several conformation-dependent epitopes of gp120/gp41 inhibit CCR-5-dependent cell-to-cell fusion mediated by the native envelope glycoprotein of a primary macrophage-tropic HIV-1 isolate

The β-chemokine receptor CCR-5 is essential for the efficient entry of primary macrophage-tropic HIV-1 isolates into CD4+ target cells. To study CCR-5-dependent cell-to-cell fusion, we have developed an assay system based on the infection of CD4+ CCR-5+ HeLa cells with a Semliki Forest virus recombinant expressing the gp120/gp41 envelope (Env) from a primary clade B HIV-1 isolate (BX08), or from a laboratory T cell line-adapted strain (LAI). In this system, gp120/gp41 of the “nonsyncytium-inducing,” primary, macrophage-tropic HIV-1BX08 isolate, was at least as fusogenic as that of the “syncytium-inducing” HIV-1LAI strain. BX08 Env-mediated fusion was inhibited by the β-chemokines RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory proteins 1β (MIP-1β) and by antibodies to CD4, whereas LAI Env-mediated fusion was insensitive to these β-chemokines. In contrast soluble CD4 significantly reduced LAI, but not BX08 Env-mediated fusion, suggesting that the primary isolate Env glycoprotein has a reduced affinity for CD4. The domains in gp120/gp41 involved in the interaction with the CD4 and CCR-5 molecules were probed using monoclonal antibodies. For the antibodies tested here, the greatest inhibition of fusion was observed with those directed to conformation-dependent, rather than linear epitopes. Efficient inhibition of fusion was not restricted to epitopes in any one domain of gp120/gp41. The assay was sufficiently sensitive to distinguish between antibody- and β-chemokine-mediated fusion inhibition using serum samples from patient BX08, suggesting that the system may be useful for screening human sera for the presence of biologically significant antibodies.

Antibodies to ribosomal P proteins of Trypanosoma cruzi in Chagas disease possess functional autoreactivity with heart tissue and differ from anti-P autoantibodies in lupus

Anti-P antibodies present in sera from patients with chronic Chagas heart disease (cChHD) recognize peptide R13, EEEDDDMGFGLFD, which encompasses the C-terminal region of the Trypanosoma cruzi ribosomal P1 and P2 proteins. This peptide shares homology with the C-terminal region (peptide H13 EESDDDMGFGLFD) of the human ribosomal P proteins, which is in turn the target of anti-P autoantibodies in systemic lupus erythematosus (SLE), and with the acidic epitope, AESDE, of the second extracellular loop of the β1-adrenergic receptor. Anti-P antibodies from chagasic patients showed a marked preference for recombinant parasite ribosomal P proteins and peptides, whereas anti-P autoantibodies from SLE reacted with human and parasite ribosomal P proteins and peptides to the same extent. A semi-quantitative estimation of the binding of cChHD anti-P antibodies to R13 and H13 using biosensor technology indicated that the average affinity constant was about 5 times higher for R13 than for H13. Competitive enzyme immunoassays demonstrated that cChHD anti-P antibodies bind to the acidic portions of peptide H13, as well as to peptide H26R, encompassing the second extracellular loop of the β1 adrenoreceptor. Anti-P antibodies isolated from cChHD patients exert a positive chronotropic effect in vitro on cardiomyocytes from neonatal rats, which resembles closely that of anti-β1 receptor antibodies isolated from the same patient. In contrast, SLE anti-P autoantibodies have no functional effect. Our results suggest that the adrenergic-stimulating activity of anti-P antibodies may be implicated in the induction of functional myocardial impairments observed in cChHD.

The epitopes for some antiphospholipid antibodies are adducts of oxidized phospholipid and β2 glycoprotein 1 (and other proteins)

Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as β2 glycoprotein 1 (β2GP1). Addition of human β2GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of β2GP1, polylysine, or low-density lipoprotein to be a “cofactor” for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of β2GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-14C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-14C]-PC, led to formation of covalent adducts with β2GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with β2GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including β2GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.

Homodimerization of tumor-reactive monoclonal antibodies markedly increases their ability to induce growth arrest or apoptosis of tumor cells

Monoclonal antibodies (mAbs) that exert antitumor activity can do so by virtue of their effector function and/or their ability to signal growth arrest or cell death. In this study, we demonstrate that mAbs which have little or no signaling activity—i.e., anti-CD19, CD20, CD21, CD22 and Her-2—can become potent antitumor agents when they are converted into IgG–IgG homodimers. The homodimers exert antigrowth activity by signaling G0/G1 arrest or apoptosis, depending upon which cell surface molecule they bind. This activity is specific and, in the case of the anti-CD19 mAb, did not require an Fc portion. These results offer the possibility that homodimers of other tumor-reactive mAbs which have little antitumor activity as monomers might be potent, antitumor agents.

Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 ± 1) × 10−11 M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.

Improved cervical smear assessment using antibodies against proteins that regulate DNA replication

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

A retro-inverso peptide corresponding to the GH loop of foot-and-mouth disease virus elicits high levels of long-lasting protective neutralizing antibodies

Peptides corresponding to the immunodominant loop located at residues 135–158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141–159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-d retro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the corresponding l-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the corresponding l-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines.

A Fungal Kinesin Required for Organelle Motility, Hyphal Growth, and Morphogenesis

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

In situ detection of activated Bruton’s tyrosine kinase in the Ig signaling complex by phosphopeptide-specific monoclonal antibodies

Bruton’s tyrosine kinase (Btk) is a critical transducer of signals originating from the B cell antigen receptor (BCR). Dosage, sequential phosphorylation, and protein interactions are interdependent mechanisms influencing Btk function. Phosphopeptide-specific mAbs recognizing two distinct phosphotyrosine modifications were used to quantify Btk activation by immunofluorescent techniques during B cell stimulation. In a population of cultured B cells stimulated by BCR crosslinking and analyzed by flow cytometry, transient phosphorylation of the regulatory Btk tyrosine residues (551Y and 223Y) was detected. The kinetics of phosphorylation of the residues were temporally distinct. Tyrosine 551, a transactivating substrate site for Src-family kinases, was maximally phosphorylated within ≈30 seconds of stimulation as monitored by flow cytometry. Tyrosine 223, an autophosphorylation site within the SH3 domain, was maximally phosphorylated at ≈5 minutes. Btk returned to a low tyrosine phosphorylation level within 30 minutes, despite persistent elevation of global tyrosine phosphorylation. Colocalization of activated Btk molecules with the crosslinked BCR signaling complex was observed to coincide with the period of maximal Btk tyrosine phosphorylation when stimulated B cells were analyzed with confocal microscopy. The results of these in situ temporal and spatial analyses imply that Btk signaling occurs in the region of the Ig receptor signaling complex, suggesting a similar location for downstream targets of its activity.

Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.