Biomedical subjects
C Milstein
Publications and source records attributed to C Milstein.
Targeting of non-Ig sequences in place of the V segment by somatic hypermutation.
Affinity maturation of antibodies is characterized by localized hypermutation of the DNA around the V segment. Here we show, using mice containing single or multiple transgene constructs, that an immunoglobulin V kappa segment can be replaced by human beta-globin or prokaryotic neo or gpt genes without affecting the rate of hypermutation; the V gene itself is not necessary for recruiting hypermutation. The ability to target hypermutation to heterologous genes in vivo could find more general applications in biology.
A reporter gene to analyse the hypermutation of immunoglobulin genes.
The affinity maturation of antibodies is driven by somatic hypermutation which is localized to specific segments of the coding genes. The information available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fusion gene between a kappa chain and a selectable neomycin resistance gene, neor. The neor gene, which includes the SV40 small t intron and polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged V kappa OX1-J kappa 5 gene. Expression of neor activity is therefore dependent on the upstream immunoglobulin sequence. A stop codon was placed in the CDR1 region so that only mutants survive treatment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating spontaneous mutants. Neomycin-resistant clones arose at an estimated rate of 1 x 10(-8)/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-frame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting all the sequences downstream of the J kappa 5 with others known to be required for full hypermutation in vivo. Different cell lines were transfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (single base addition and deletion plus two point mutations) was detected. The reporter gene system therefore seems suitable to test culture conditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.
Universal cloning and direct sequencing of rearranged antibody V genes using C region primers, biotin-captured cDNA and one-side PCR.
Polymerase chain reaction (PCR) cloning has greatly facilitated the cloning of heavy and light chain genes from B cells and hybridomas and has been critical for the generation of natural antibody gene libraries for expression in bacteria and on filamentous phages. There remain difficulties, however, in cloning VH and VL genes from a number of mouse and rat hybridoma lines and from B cells from several other species due to insufficient sequence information. Here we describe a rapid and 'universal' strategy for cloning rearranged antibody genes from any species for which the sequence of the C segment(s) are known. First strand synthesis is primed with a biotinylated C region primer and full length cDNA is captured on streptavidin-coated magnetic beads for tailing with dGTP and terminal deoxynucleotidyl transferase. After tailing, the cDNA is captured again, amplified using polyC primers and used for direct sequencing or cloning. The use of C region primers and cDNA capture ensures that this one-side PCR procedure is efficient and rapid as well as being entirely independent of the sequence of the V segment. We demonstrate its application to the direct sequencing or cloning of the H and L chain genes from six mouse and rat hybridomas and propose that the method described will find applications in three areas: (i) cloning rearranged antibody genes in all cases in which cloning with V-J primers is not possible; (ii) repertoire studies in which an unbiased cloning procedure is required for accurate estimate of gene usage; and (iii) generation of VH and VL gene libraries from immunised animals.
Somatic hypermutation.
For the generation of secondary response antibodies, immunoglobulin genes are subjected to hypermutation. Cells expressing antibodies with higher affinity are then selected by antigen. Recent clues to the mechanism of hypermutation come from experiments using transgenic mice enabling analysis of the controlling cis-acting elements and the intrinsic features of the hypermutation, dissociated from the effects of antigenic selection.
A general model of drug prescription.
Currently, there is no widely accepted structured representation of drug prescription. Nevertheless, a structured representation is required for entering and storing drug prescriptions avoiding free text in computerized systems, and for drug prescription reviews. Derived from part of the work of the European OPADE project, we describe an object-oriented model of drug prescription which incorporates important concepts such as the phase and triggering event concepts. This model can be used to record all drug prescriptions, including infusions, in a structured way avoiding free text. The phase concept allows the storage of sequentially ordered dosage regimens for a drug within the same prescription. The prescription triggering event concept allows recording of the administration of a drug conditional to dates, symptoms and clinical signs, medical procedures, and everyday life events. This model has been implemented within the OPADE project; the corresponding aspects of the user interface are presented to show how this model can be used in practice. Even if other new attributes may be added to the described objects, the structure of this model is suitable for general use in software which requires the entry, storage and processing of drug prescriptions.
Modeling drug information for a prescription-oriented knowledge base on drugs.
There exists little theoretical analysis how to represent knowledge on drugs required for computerized drug-prescription applications. A work package drug information modeling is described which was part of the European OPADE project. We describe the content and structure of a Drug Knowledge Base (DKB) designed to meet the requirements of decision-support systems in the domain of drug therapy, and to facilitate data transfer from various information sources. The definition of the DKB content is derived from the analysis of information requirements at the various stages of the process of the clinical usage of drugs (prescribing, administration, and follow-up). The DKB structure results from the classification of the various data items along two dimensions: (1) entities in the pharmaco-therapeutic domain for which information must be defined (the PharmacoTherapeutic Group, the Component, the ManufacturedPreparation, and the Presentation), and (2) the validity score of the pharmaco-therapeutic information (international, national, or local).
Somatic mutation of immunoglobulin lambda chains: a segment of the major intron hypermutates as much as the complementarity-determining regions.
The rate and nature of hypermutation of immunoglobulin genes are of prime importance in the affinity maturation of antibodies. Although a considerable body of information has been gathered for kappa light chains, there is much less data for lambda chains. We have derived a large data base of somatic mutants of mouse lambda 1 light chains from Peyer's patches germinal center B cells. The endogenous lambda 1 genes mutate at a rate comparable to that previously found for a kappa transgene (V kappa ox1). There are intrinsic hot spots of mutation common to both in-frame and out-of-frame rearrangements; these hot spots cluster in hypermutating domains. In contrast to the pattern seen for V kappa Ox1, the hot spot clusters are found not only in complementarity-determining region (CDR)1 but also in CDR2 and CDR3; mutations also cluster in the joining/constant region intron. The differences between the pattern of mutations in V kappa Ox1 and lambda 1 light chains are discussed.
Conformational isomerism and the diversity of antibodies.
The fact that one cell encodes a single antibody sequence does not necessarily mean that the resulting antibody folds into a single structure, although this is a common assumption. Here we challenge this view and suggest that many antibodies do not have a single conformation at the combining site. The basis for this proposal comes from the kinetic analysis of a set of murine hybridomas derived from defined stages of the immune response to 2-phenyl-5-oxazolone (Ox). Among them we have identified three antibodies that exhibit complex hapten-binding kinetics. We observed biphasic or triphasic reactions in stopped-flow fluorescence experiments, indicating that ligand binding involved isomerization, as well as associative steps. The existence of an equilibrium between at least two antibody conformations, with ligands binding preferentially to one form, was deduced from the variation with hapten concentration of the apparent rate of each phase.
Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing.
We have previously reported down-regulation of mRNA expression of some of the kappa light chain transgenes in a hybridoma derived from a secondary immune response. Of the five heavily mutated transgene copies present in that hybridoma, three included premature stop codons and were poorly represented at the mRNA level. Here we show that the nonsense mutations are the cause of the low mRNA levels. While we found no evidence that the reduction in mRNA abundance was attributable to an increased rate of cytoplasmic mRNA decay, the amount of cytoplasmic mRNA correlated with the accumulation of unspliced transcripts in the nucleus. Similar results were obtained with a chimeric immunoglobulin gene containing a premature chain termination codon in the variable gene segment. We suggest that inhibition of splicing induced by in-frame premature stop codons is an important mechanism for down-regulation of undesirable immunoglobulin transcripts.
Alternative splicing generates secretory isoforms of human CD1.
Human CD1 genes are a family of five non-polymorphic genes that, although homologous to both class I and II major histocompatibility complex genes, map to chromosome 1. Only three of the antigens, CD1a, -b, and -c, have been clustered with monoclonal antibodies. They are noncovalently associated with beta 2-microglobulin and may function as nonclassical antigen-presenting molecules. Here we analyze their expression in mouse myeloma transfectants and human thymocytes and find mRNA splicing complexity. This manifests itself as incomplete splicing, alternative splicing, utilization of cryptic splice sites, and the generation of alternative reading frames. In the case of CD1A transfectants, we demonstrate that the major protein product is secreted and show by amino acid sequence analysis that this is derived from an unspliced transcript. A second major CD1a component appears to be retained intracellularly. The production of alternatively spliced transcripts in the thymus is not a feature of all CD1 genes. Although in the case of CD1A only the transcript encoding the cell surface CD1a isoform is found, CD1C and -E produce complex intrathymic splicing patterns. The CD1C transcripts predict the expression of a secreted CD1c isoform in the human thymus, which we detect in CD1C transfectant culture supernatants. CD1 gene expression is thus characterized by considerable splicing complexity, and the difference between the splicing patterns found in different environments suggests that this is tissue specific.
Elements regulating somatic hypermutation of an immunoglobulin kappa gene: critical role for the intron enhancer/matrix attachment region.
Following encounter with antigen, the immunoglobulin genes in B lymphocytes undergo somatic hypermutation. Most nucleotide substitutions are introduced into a region flanked by the V gene promoter and intron enhancer. Experiments described here using transgenic mice revealed that the V kappa promoter does not contain specific signals since hypermutation was retained on substituting it by a beta-globin promoter. However, both the kappa intron and kappa 3' enhancer regions were found to be essential for full hypermutation. This dependence of hypermutation on both enhancers contrasts with transgene expression in hybridomas in which only the 3' enhancer (and not the intron enhancer) is necessary to achieve high mRNA levels. The results show that full hypermutation depends on multiple elements, removal of some of which may drastically impair but not totally abolish the process.
The 5' boundary of somatic hypermutation in a V kappa gene is in the leader intron.
The maturation of the immune response involves the hypermutation of antibody genes and the selection of B cells expressing receptors with improved antigen binding properties. Somatic hypermutation of antibody genes is targeted to a small region approximately 1 kb surrounding the rearranged V gene. The precise definition of the 5' limit is not yet clear since the data base of somatic mutations upstream of the V region is very restricted. The available data suggest that it lies close to the promoter region and this has been used to implicate transcription in the mechanism leading to hypermutation. Here we present an extensive analysis of mutations in the 5' region of a single kappa light chain gene. A large data base from highly mutated sequences was obtained from anti-oxazolone hybridomas expressing the V kappa Ox1-J kappa 5 light chain and from polymerase chain reaction-derived clones from splenic and Peyer's patches of transgenic mice expressing the same V kappa Ox1-J kappa 5 gene combination. Although mutations were found in the 5'-flanking segment, the rate of mutation in the V-J segment was about 20-fold higher. A sharp decline between those two mutation rates is evident but the boundary was found in the leader intron of the V kappa Ox1 gene, about 150 bases downstream of the initiation of transcription site.
Age-related decrease in the proportion of germinal center B cells from mouse Peyer's patches is accompanied by an accumulation of somatic mutations in their immunoglobulin genes.
Somatic hypermutation of immunoglobulin genes and the generation of memory B cells seems to take place in germinal centers, which are chronically present in Peyer's patches. Age-associated changes in the germinal center B cell compartment of Peyer's patches and in the mutations of a kappa light chain transgene were analyzed in unimmunized mice. Somatic mutations accumulate in germinal center B cells slowly and continuously to reach an apparent plateau when the animals are around 5 months old. In contrast, the proportion of germinal center B cells reaches a maximum in very young mice (about 2 months old) and decreases progressively thereafter. These results suggest that the highly mutated B cells in older mice arise by the successive accumulation of mutations in memory cells. The data also show that the optimum time for the analysis of hypermutation of transgenes in Peyer's patches is when the mice are about 5 months old.
OPADE: optimization of drug prescription using advanced informatics.
The computerized drug prescription system (CDPS) is an old, almost obsolete, dream, and numerous researchers have developed prototypes or even full fledged packages. But few systems are currently used in clinical practice. The OPADE project, presented in this paper, aims at developing a computerized system which allows for optimization of drug prescription from a medical, patient compliance and economical point of view; the system will be adapted to different European countries, integrated and customized to local medical practice so as to insure user acceptance.
Monoclonal antibodies in hematology.
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Transcriptional regulation of HLA-A and -B: differential binding of members of the Rel and IRF families of transcription factors.
HLA-A and -B transplantation antigens can be expressed differentially at the basal level and in response to interferons (IFNs). To determine which DNA control elements and nuclear factors are responsible for these differences, HLA-A and -B upstream regulatory regions were used in expression and mobility-shift analyses. The HLA-A enhancer was found to contain two Rel (KBF/NF-kappa B) binding motifs, while the HLA-B enhancer has only one and is transactivated less well by overexpression of the NF-kappa B p65 subunit. On the other hand, the HLA-B IFN response element mediates a much stronger induction by IFNs and has a higher affinity for IRF-1 and -2, which are transcription factors implicated in the regulation of major histocompatibility complex class I genes. These results suggest a molecular basis for the way in which HLA-A and -B loci have adapted to be differentially expressed and to respond to different sets of cytokine signals.
Analysis of somatic hypermutation in mouse Peyer's patches using immunoglobulin kappa light-chain transgenes.
We have exploited mice transgenic for an immunoglobulin kappa light chain in order to show that immunoglobulin genes in the B cells of Peyer's patches in unimmunized mice carry a high level of somatic mutations. Most of the mutations are found in the subpopulation of B cells which, based on peanut agglutinin binding, derive from the germinal centers. The number of mutations per clone and their distribution along the variable gene segment (indicative of untemplated point mutations) are very similar to those found in antigen-specific splenic B cells of normal mice after secondary immunization. The mutations accumulate mainly in complementarity-determining region 1, in particular in some specific codons (Ser-26, Ser-31, and Ser-77) which have been previously recognized as intrinsic hypermutational hotspots. These results suggest that, as in the spleen, somatic mutation occurs in B cells which have migrated to the germinal centers, probably as a consequence of stimulation by antigens present in the gut environment. Transgenic animals are increasingly being used to define the signals involved in hypermutation. However, their subsequent study is very time-consuming because it is based on immunization and analysis of hybridomas or antigen-selected cells. We propose that the use of Peyer's patches of unimmunized adult mice offers a reliable and simple approach to analyze hypermutation of transgenes.