Molecular characterization of a cross-reactive idiotope on human immunoglobulins utilizing the VH4-21 gene segment.

The anti-idiotypic (anti-Id) antibody (Ab) 9G4 binds a cross-reactive idiotope (CRI) present in a select group of human autoantibodies. This Id has been localized to the portion of immunoglobulin (Ig) heavy (H) chains encoded by the VH4-21 gene segment, a member of the human VH4 family. This gene segment is utilized by essentially all cold agglutinin (CA) Abs with I/i specificity isolated from patients with CA disease stemming from chronic lymphoproliferative disorders. In this study, mutational analysis of a CA has been used to determine the structural basis for 9G4 binding to Abs utilizing the VH4-21 gene segment. Recombinant CA H chain mutants were produced and their 9G4 reactivity determined. Mutants were generated by exchanging VH4-21 sequences in the FR1, CDR1, and CDR2 with corresponding sequences from a closely related gene segment V71-2, a VH4 family member that is associated neither with Abs having CA activity nor with Abs that react with 9G4. The results indicate that the motif AVY at amino acid positions 23-25 in FR1 defines the 9G4 idiotope. Reaction of these recombinant Abs with a polyclonal rabbit anti-CA antiserum absorbed to render it specific for a CA CRI also maps predominantly to FR1. These findings indicate that the solvent-exposed FR1 plays an important role in eliciting an immune response to Igs.

Pharmacological inhibitors of NF-kappaB accelerate apoptosis in chronic lymphocytic leukaemia cells.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a critical role in the inappropriate survival of various types of malignant cells. Chronic lymphocytic leukaemia (CLL) is the most common B-cell malignancy in the Western world. Although overexpression and regulation of NF-kappaB has been described in CLL, its function remains unclear. Exposure of CLL cells to BAY117082 or Kamebakaurin, potent pharmacological inhibitors of the NF-kappaB pathway, accelerated apoptosis in approximately 70% of cases. Sensitivity to NF-kappaB pathway inhibitors was not related to the prognostic markers VH status, CD38 or Zap70 expression, or to the levels of nuclear NF-kappaB. Normal peripheral B cells were resistant to the apoptosis-inducing effects of these compounds. Cell death induced by the inhibitors was associated with activation of caspase-9 and -3, and loss of mitochondrial membrane polarization, but did not involve changes in the expression of Bcl-2 or Mcl-1. Inhibitors caused an increase in c-jun NH2-terminal kinase activity in CLL, but this did not appear to be important for apoptosis. Microarray analysis identified some potential novel NF-kappaB target genes, including interleukin-16- and the Bcl-2- related survival protein Bcl-w. These results demonstrate that a substantial proportion of CLL are dependent on NF-kappaB for enhanced survival and suggest that inhibition of NF-kappaB may have therapeutic potential.

Immunogenetic analysis reveals that epitope shifting occurs during B-cell affinity maturation in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a liver disease characterized by serum autoantibodies against the pyruvate dehydrogenase complex (PDC) located in the inner mitochondrial membrane. The predominant target in PDC has previously been localized to the inner lipoyl domain (ILD) of the E2 subunit. The etiology of PBC is unknown, although molecular mimicry with bacterial PDC has been proposed. Here, we have investigated the etiology of PBC and nature of the autoimmune response by analyzing the structure of a human monoclonal antibody with ILD specificity. Mutants of the monoclonal antibody, which was originally isolated from a patient with PBC, were expressed as Fab by phage display, and tested for reactivity against recombinant domains of the E2 subunit. Fab in which the V(H)-encoded portions were reverted to germline lost reactivity against the ILD alone, but recognized a different epitope in a didomain construct encompassing the ILD, hinge region and E1/E3 binding domain. The complete V(H) and V(L )germline revertant was unreactive with the human ILD and didomain, the Escherichia coli didomain, and whole PDC. We hypothesize that the IgM on the surface of the naïve B-cell first recognizes an as yet unidentified antigen, and that accumulation of somatic mutations results in an intermolecular epitope shift directed towards an epitope involving the E1/E3 binding domain. Further mutations result in the specificity being redirected to the ILD. These findings also suggest that bacterial molecular mimicry is not involved in initiating disease.

The I binding specificity of human VH 4-34 (VH 4-21) encoded antibodies is determined by both VH framework region 1 and complementarity determining region 3.

Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.

Anti-idiotypic antibody D12 and superantigen SPA both interact with human VH3-encoded antibodies on the external face of the heavy chain involving FR1, CDR2 and FR3.

The mouse monoclonal antibody (mAb) D12 specifically binds in the variable region (idiotype) of human V(H)3 encoded antibodies. We used mutational analysis to determine the subregions of a V(H)3 encoded antibody which effect the interaction with mAb D12. Recombinant antibodies composed of mutant heavy chains were produced using the baculovirus expression system. The results of this topographical study indicate that the combined conformations of FRI, CDR2 and FR3 are critical for mAb D12 binding. MAb D12 binding was not effected either by the heavy chain CDR3 sequence nor by the light chain. We previously demonstrated that structures within the same three subregions are required for the B cell superantigen Staphylococcal protein A (SPA) binding to V(H)3 encoded antibodies. Thus, some anti-idiotypic antibodies can interact with antibodies in a similar fashion to superantigens.

Learning-related fos-like immunoreactivity in the chick brain: time-course and co-localization with GABA and parvalbumin.

Previous work has shown that, after domestic chicks have learned the characteristics of an object (visual imprinting), there is a learning-related increase in the numerical density of Fos-immunopositive neurons in the intermediate and medial part of the hyperstriatum ventrale, a forebrain region that is a site of recognition memory for the imprinted object. The present study describes the time-course of this effect and has used double-labelling immunocytochemistry to identify neuronal types in which the effect occurs. Chicks were trained by exposure for 1 h to an imprinting (training) stimulus and then given a preference test to determine the strength of imprinting (i.e. of learning). Strongly imprinted chicks were killed 2, 2.5, 3, 3.5 or 4 h (12 chicks in each group) after the start of training and a further group of 12 chicks remained untrained. Sections from the chicks' brains were stained for Fos-like immunoreactivity, and the numerical density of Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale was counted. Relative to untrained chicks, there was a 60% increase in the number of Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale 2 h after the start of training (P = 0.02), but not at any other time. Sections from 10 trained chicks, two killed at each of the above times after training, and from two untrained chicks were stained with anti-Fos antibody as before and also with an antibody against GABA. Approximately 95% of the Fos-positive neurons in the intermediate and medial part of the hyperstriatum ventrale were also immunopositive for GABA. In neurons immunopositive for GABA, there were significantly (P = 0.02) more Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale 2 h after the start of training than in untrained chicks. Five chicks killed 2 h after training and five untrained chicks yielded sections for the next experiment; sections were double labelled for (i) Fos and (ii) either Calbindin-D28k or parvalbumin. Training gave rise to a significant (P = 0.017) increase in numerical density of Fos-positive nuclei of neurons that were immunonegative for Calbindin-D28k. This increase occurred in neurons that were immunopositive for parvalbumin. The use of alternative antibodies for GABA, Calbindin-D28k and parvalbumin in trained and untrained chicks confirmed the double-staining pattern observed in the quantitative experiments. The results demonstrate that the learning-related increase in Fos-like immunoreactivity following training is transitory and have localized the increase to a population of neurons immunopositive for GABA and parvalbumin, but not Calbindin-D28k.

Antibody production in the baculovirus expression system.

The proven ability of insect cells to produce murine and human antibodies renders the baculovirus system amenable to the synthesis of both naturally occurring antibodies and designed mutants. Study of these antibodies should provide a basis for rational antibody design useful in human therapy.

Staphylococcal protein A binding to VH3 encoded immunoglobulins.

Staphylococcal protein A (SPA) is a B-cell superantigen which binds specifically to the variable region of human VH3 encoded antibodies. We undertook to identify the VH3 regions involved in the interaction with SPA by producing mutant antibodies in the baculovirus expression system. We had previously shown that a single amino acid change at position 57 in the CDR2 of a human SPA nonbinding VH3 encoded rheumatoid factor converted it to an SPA binder, implicating CDR2 in SPA binding. When regions of the mutated binder were exchanged with those from a mouse nonbinding antibody, the pattern of SPA binding indicated that residues in FR1, CDR2 and FR3 are involved in the interaction between VH3 encoded antibodies and SPA. In addition, all three regions are simultaneously required for SPA binding to occur. When any one of the three regions was altered, SPA binding was severely disrupted.

A human monoclonal antibody encoded by the V4-34 gene segment recognises melanoma-associated ganglioside via CDR3 and FWR1.

A heterohybridoma cell line producing the human monoclonal antibody (MoAb) MDT.1 has been established. The heavy chain of MoAb MDT.1 is encoded by the VH gene segment V4-34 (previously designated VH4-21), and the light chain is encoded by the V kappa 1-L12a gene segment, both in germline configuration. MDT.1 has reactivity against lipid A, double- and single-stranded DNA, red blood cell associated i antigen, and ganglioside antigens. In a panel of tumour cell lines, MDT.1 reacted specifically with melanoma cells and other tumour cells of neuroectodermal origin. Cellular recognition appears to be via tumour-associated ganglioside antigens, and may involve the minimal essential epitope NeuNac alpha 2-->3Gal beta 1-->-4Glc-. Binding to ganglioside antigen is inhibited by the monoclonal anti-idiotypic antibody 9G4. Since the 9G4 idiotope is located in framework region 1 (FWR1) of V4-34-encoded antibodies, this region is likely to be involved, either directly or indirectly, in ganglioside binding. The complementarity-determining region 3 (CDR3) of MDT.1 is arginine rich, with five out of 12 residues being arginine and these residues are candidates for interaction with the negatively charged ganglioside. The ability of MoAb MDT.1 to recognise ganglioside antigens is associated with potentially useful anti-tumour activity.

A role for eukaryotic initiation factor 4B overexpression in the pathogenesis of diffuse large B-cell lymphoma.

Dysregulated expression of factors that control protein synthesis is associated with poor prognosis of many cancers, but the underlying mechanisms are not well defined. Analysis of the diffuse large B-cell lymphoma (DLBCL) translatome revealed selective upregulation of mRNAs encoding anti-apoptotic and DNA repair proteins. We show that enhanced synthesis of these proteins in DLBCL is mediated by the relief of repression that is normally imposed by structure in the 5'-untranslated regions of their corresponding mRNAs. This process is driven by signaling through mammalian target of rapamycin, resulting in increased synthesis of eukaryotic initiation factor (eIF) 4B complex (eIF4B), a known activator of the RNA helicase eIF4A. Reducing eIF4B expression alone is sufficient to decrease synthesis of proteins associated with enhanced tumor cell survival, namely DAXX, BCL2 and ERCC5. Importantly, eIF4B-driven expression of these key survival proteins is directly correlated with patient outcome, and eIF4B, DAXX and ERCC5 are identified as novel prognostic markers for poor survival in DLBCL. Our work provides new insights into the mechanisms by which the cancer-promoting translational machinery drives lymphomagenesis.

Immunoglobulin sequence analysis and prognostication in CLL: guidelines from the ERIC review board for reliable interpretation of problematic cases.

Immunoglobulin gene sequence analysis is widely utilized for prognostication in chronic lymphocytic leukemia (CLL) and the definition of standardized procedures has allowed reliable and reproducible results. Occasionally, a straightforward interpretation of the sequences is not possible because of the so-called 'problematic sequences' that do not fit the 'classic' interpretation and pose scientific questions at the cross-road between hematology and immunology. Thanks to a dedicated effort within the European Research Initiative on CLL (ERIC), we have now the possibility to present such cases, offer a scientific explanation and propose recommendations in terms of prognostication.

Molecular characterization of cold agglutinins.

An unusual bias involving the exclusive usage of the V4-34 gene segment by pathogenic antibodies with I/i antigen specificity has been documented in the literature. In addition, all unmutated and several mutated V4-34 encoded antibodies have been shown to be reactive with the anti-idiotypic monoclonal antibody 9G4. The 9G4 Id, therefore, is a marker for V4-34 gene segment expression. Based on these two correlations, it became vital to localize and characterize the nature of the 9G4 Id and to determine the relationship between the Id and I binding. Mutational analysis indicated that the 9G4 Id is located in framework region 1 (FR1) of V4-34 encoded antibodies. Two distinct sections of FR1, encompassing amino acid residues 6-12 and 23-25, form the 9G4 Id. Mutational analysis demonstrated that both FR1 and CDRH3 were required for I binding. When either one was disrupted, the mutant antibody could not bind I. This indicates that I binds through a framework region, and not exclusively through CDRH3. This renders the I interaction with the V4-34 encoded portion of immunoglobulins unconventional, with characteristics similar to superantigen binding to immunoglobulin through FR. When the FR1 DNA sequence of V4-34 was exchanged for FR1 sequences from other VH families I binding was lost, providing a structural explanation for this restricted VH usage. An understanding of the localization and structure of the 9G4 Id and the requirements of V4-34 encoded antibodies for I binding provide insights into the structure of pathogenic antibodies and their requirements for binding antigen. This information should be useful in analyzing new interactions such as the lytic activity of some V4-34 encoded antibodies for B cells.

A DNA analysis program designed for computer novices working in an industrial-research environment.

A program designed for DNA sequence analysis by people with no computer background or experience is described. The criteria used in the program design were (i) user friendliness, (ii) ability to handle large DNA files (greater than 10 kilobases), (iii) low storage requirements so that it could be put on a personal computer (IBM), (iv) written in a widely used language (FORTRAN 77) and (v) ability to handle all of the needed tasks for DNA analysis. The program consists of an executive that controls a set of modular subprograms. A file management system using 'SEQ' and 'SITE' files is also in place. SEQ files contain long DNA nucleotide sequences (e.g. restriction enzyme sites). Related SEQ and SITE files are grouped into categories. A SITE category can include related sequences a user wishes to search for in a longer SEQ sequence from a designated category. For example, all of the restriction enzyme recognition sites can group into a single category called 'Enzymes' and can be searched for in a SEQ sequence which has been placed in the SEQ file category 'Plasmids'. Colour graphics is also used as a visual aid.

Methylation blockage and other improvements to a comprehensive DNA analysis program.

A comprehensive DNA analysis computer program was described in the second special issue of Nucleic Acids Research on the applications of computers to research on nucleic acids by Stone and Potter (1). Criteria used in designing the program were user friendliness, ability to handle large DNA sequences, low storage requirement, migratability to other computers and comprehensive analysis capability. The program has been used extensively in an industrial-research environment. This paper talks about improvements to that program. These improvements include testing for methylation blockage of restriction enzyme recognition sites, homology analysis, RNA folding analysis, integration of a large DNA database (GenBank), a site specific mutagenesis analysis, a protein database and protein searching programs. The original design of the DNA analysis program using a command executive from which any analytical programs can be called, has proven to be extremely versatile in integrating both developed and outside programs to the file management system employed.

The molecular specificity of insulin autoantibodies.

Insulin autoantibodies (IAA) are one of several markers for Type I (autoimmune) diabetes, but alone deserve special attention. Unlike the other markers, their ligand is unique to the beta cell. IAA are the first markers to appear during the symptomless period which precedes diabetes and they are present in the vast majority of young children destined to develop diabetes. The primary and tertiary structures of insulin have been known for decades. Binding studies with insulin variants have shown epitope restriction that can distinguish Type 1 diabetes-predictive from non-predictive IAA-positive sera, thereby improving specificity for the test. With two major international Type 1 diabetes prevention trials underway, there is a pressing need to refine markers that reliably indicate the presence of, and remission from, autoimmune insulitis. The binding regions of antibodies are assembled from three multi-gene families, and some of their diversity derives from random mutation during their antigen-driven maturation. There is evidence that mature IAA derive from germline-encoded 'natural' antibodies, and that the gene segments utilised by IAA may be influenced by clinical context. Monoclonal anti-idiotypic (anti-Id) antibodies can serve as probes for antibody variable region determinants, and antibodies to the different epitopes of beef and porcine insulins have already been analysed with monoclonal reagents. Used as antibodies in a radioimmunoassay format, monoclonal anti-Ids will identify and measure autoantibody idiotopes as if they were ligands. The challenge now is to replace the conventional radiobinding assays for IAA, which only detect and titrate, with radioimmunoassays that can be standardised in absolute units. There is sufficient evidence for the existence of Type 1 diabetes-predictive IAA idiotopes to justify the development of idiotope-specific radioimmunoassays which ignore Type 1 diabetes-unrelated IAA.

Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells.

A comparison of the ability of vesicular stomatitis virus (VSV) to generate and replicate defective interfering (DI) particles in primary chick embryo (CE) and mouse L cells was investigated as a means of analyzing host control over DI-particle synthesis and interfering capacity. Serial undiluted passage of VSV in CE and L cells indicate that VSV-DI particles are generated and (or) replicate with greater efficiency in CE than in L cells. When DI particles accumulate in L cells, they are able to interfere with infectious particle replication. The DI particles from CE cells interfered to the same extent with infectious particle replication in both CE and L cells. L cells, therefore, are not considered 'low-interference' hosts in which DI particles are produced and do not interfere with infectious virus replication, but rather hosts which restrict the production of DI particles.

Anti-DNA antibodies--structure and function.

Expression of monoclonal anti-DNA antibodies in vitro can be used to study the relationships between molecular structure, binding properties and pathogenicity. Bacterial and yeast systems can be used to produce antibody fragments such as Fab. The yields are potentially sufficient to allow structural studies such as crystallization, but purification of the anti-DNA Fab from the bacterial periplasm may be challenging. Mammalian cell expression systems produce lower yields, but the products are whole antibodies, which can be used in assays of pathogenicity. This article describes some recent experiments in which bacterial and mammalian systems were used to study human monoclonal anti-DNA antibodies. Light chain sequence motifs were found to be important both in binding to antigens and in determining pathogenicity of the antibodies in severe combined immunodeficiency mice. The distribution of B cell subpopulations is disturbed in patients with systemic lupus erythematosus (SLE). These patients, like those with infectious mononucleosis, have an overall B cell lymphopenia but an increased frequency of plasmablasts/early plasma cells in their blood. Some of these early plasma cells belong to clones that have rearranged the V(H) gene V4-34. There is a selective rise in immunoglobulins encoded by this gene in both infectious mononucleosis and SLE.

Modification of Trichoplusia ni nuclear polyhedrosis virus passaged in vivo.

A designated strain of plaque-purified Trichoplusia ni nuclear polyhedrosis virus (NPV) was used to initiate a serial passage series in vivo. The virulence of polyhedra inclusion bodies (PIB) and the distribution of MP (many PIB) and FP (few PIB) strains in hemolymph were monitored. When virus was passaged per os as PIB, there was no major change in virulence after 15 passes in insects. Plaque analysis of hemolymph from the infected insects indicated that there was no selection for the FP strain of virus. There was little overall change in the infectivity of nonoccluded virus (NOV) after 16 passes by injection into T. ni larvae; however, plaque analysis of the hemolymph from the infected insects indicated progressive selection of FP virus. It is postulated that when PIB are the vehicles of virus transmission, there is no progressive accumulation of noninfectious FP PIB; however, when NOV are the vehicles of virus transmission, FP NOV production displaces MP virus synthesis. This may result from a growth advantage of the FP NOV over the MP NOV as shown in vitro and from the noninfectious nature of FP PIB.

Strain selection during serial passage of Trichoplusia in nuclear polyhedrosis virus.

Two strains of a nuclear polyhedrosis virus (NPV) of Trichoplusia ni were isolated on the basis of plaque morphology. They are designated as MP (having greater than 30 polyhedra per nucleus) and FP (having fewer than 10 polyhedra per nucleus). Serial, undiluted passage of plaque, purified MP nonoccluded. Virus (NOV) in tissue culture led to the production of the FP phenotype detectable at passage 9. With continued serial, undiluted passage, FP became the predominant strain. Comparative growth curves showed that FP NOV are released faster than MP NOV. MP morphology was not observed after 14 serial, undiluted passages of plaque-purified FP. By the plaque neutralization assay, NOV from both strains of virus was neutralized by the homologus and heterologous antisera. The FP phenotype was observed when FP virus was grown in culture at 17, 22, and 27 C. Hence, the FP phenotype was not considered to be the result of temperature-inhibited crystallization of polyhedrin under standard tissue culture conditions. The NOV of both strains killed insects when injected directly into the hemocoele of T. ni larvae. Only MP inclusion bodies were virulent per os. The FP inclusion bodies fed to cabbage looper larvae did not kill, and no infectious agent could be detected in the hemolymph. Electron micrographs of MP polyhedra showed bundles of nucleocapsids of normal length within the polyhedra, whereas FP polyhedra contained heterogeneous, electron-dense material, which could account for their lack of pathogenicity.

The cross-reactive idiotopes recognized by the monoclonal antibodies 9G4 and LC1 are located in framework region 1 of two non-overlapping subsets of human VH4 family encoded antibodies.

The monoclonal anti-idiotopic antibodies LC1 and 9G4 bind two non-overlapping sets of VH4 encoded antibodies. 9G4 exclusively binds VH4-21 encoded antibodies, while LC1 binds antibodies derived from VH4 family gene segments V71-2, V71-4, VH4-18, VH72-1 and V2-1. The VH4-21 gene segment is utilized by most cold agglutinin (CA) antibodies with I/i specificity, while antibodies encoded by other VH4 gene segments are associated not with CA disease, but primarily with rheumatoid-factor (RF) activity. We previously determined that the idiotope to which 9G4 binds in VH4-21-derived antibodies is located in framework region 1 (FR1). In the present study, by using mutational analysis involving individual framework- and complementarity-determining region exchanges between VH4-21- and V71-2-encoded antibodies, we have found that the idiotope to which LC1 binds in V71-2-derived antibodies also maps to FR1. The LC1 idiotope is heavy (H)-chain associated, but requires pairing with a light (L) chain for LC1 binding. Recombinant antibodies composed of a variety of kappa (kappa) and lambda (lambda) L chains paired with either a V71-2 or VH4-21 chain were produced in the baculovirus expression system. LC1 bound all of the kappa-containing antibodies but did not bind the V71-2-encoded H chain alone nor to the two lambda-containing antibodies. This experiment demonstrates that not all light chains exert equivalent influence on the conformation of the H-chain idiotope. These results indicate that the FR1 of VH4-encoded antibodies is immunogenic and suggest a physiological role of FR1 during an immune response.