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Biomedical subjects

C Milstein

Publications and source records attributed to C Milstein.

At least 37 records · Page 2Linked to original sources
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Affinity maturation leads to differential expression of multiple copies of a kappa light-chain transgene.

Transgenic animals containing rearranged heavy or light chains are used to study the process of hypermutation, which characterizes the maturation of the antibody response. LK6 mice contain five copies of a transgene coding for a light chain produced in response to the hapten 2-phenyloxazolone. We have selected hybridomas from secondary responses that express the transgene as the only light chain. Some of these hybridomas contain transgene copies carrying mutations known to improve antibody affinity. We have analysed the expression of the five transgene copies in those hybridomas. We report here that the somatic hypermutation process can affect the successful expression of antibody light-chain transgenes. When mutations that improve the antibody affinity appear in one transgene copy, antigenic selection favours cells that downregulate the other copies at multiple levels of gene expression, including examples where nonsense mutations correlate with a drop in messenger RNA level.

Amino Acid Sequence↗
PubMed1993

Passenger transgenes reveal intrinsic specificity of the antibody hypermutation mechanism: clustering, polarity, and specific hot spots.

We have analyzed somatic hypermutation in mice carrying an immunoglobulin kappa transgene in order to discriminate mutations that reflect the intrinsic specificity of the hypermutation mechanism from those highlighted by antigenic selection. We have immunized animals with three different immunogens. With one immunogen, the antigen-specific B cells express a transgenic kappa chain, which does not form part of the antibody; the transgene is a passenger free to accumulate unselected mutations. With the other two immunogens, the transgenic kappa chain constitutes the light chain of the expressed antibody. A comparison of the transgene mutations obtained under these different circumstances allows us to identify common features that we attribute to the intrinsic specificity of the hypermutation process. In particular, it yields only base substitutions and leads to hot spots occurring in individual positions (e.g., the second base of the Ser-31 codon). The mutations preferentially accumulate around the first complementarity-determining region. The process exhibits specific base substitution preferences with transitions being favored over transversions. We propose that these substitution preferences can be used to discriminate intrinsic from antigen-selected hot spots. We also note that hypermutation distinguishes between the coding and noncoding strands since pyrimidines (particularly thymidines) mutate less frequently than purines.

Amino Acid Sequence↗
PubMed1993

Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes.

Studies of the antibody hypermutation mechanism have revealed that it is not a random process but exhibits characteristic nucleotide substitution preferences. Here, Alexander Betz and colleagues show that these innate nucleotide substitution preferences can be used to examine databases of antigen-selected V gene sequences and thereby distinguish intrinsic from antigen-selected hotspots. This analysis reveals intrinsic mutational hotspots in both VH and VL genes, reflecting innate features of the hypermutation machinery which may give clues to the enzymatic mechanism.

Animals↗
PubMed1993

OPADE: development of an European computerized drug prescription system.

Many computerized drug prescription systems have been developed but they are rarely used in clinical practice because of their lack of integration with the functioning of medical institutions and the difficulty of building and maintaining a complete knowledge base on drugs. We present in this paper a system, called OPADE, which answers these shortcomings and we argue that a system actually used by practitioners may introduce a positive feed back loop in the prescribing process.

Clinical Pharmacy Information Systems↗
PubMed1992

An oscillating bubble chamber for laboratory scale production of monoclonal antibodies as an alternative to ascitic tumours.

A simple roller bottle was constructed to house three dialysis tubes, each with a capacity of 75 ml. Cells were grown inside the dialysis tubing, which was immersed in ordinary DMEM medium without serum supplement. Cultures of hybridomas at medium or low density (2 X 10(5) cells/ml) could be expanded directly in the dialysis tubes to attain a high cell density of the order of 10(7)/ml. Continuous gentle stirring of the cells was possible, since the design causes a bubble to oscillate along the length of each tube. The six cell lines tested all gave antibody concentrations of between 1.1 and 2.3 mg/ml at 20 days. Such an in vitro apparatus obviates the need to employ ascites production, because it is as simple or simpler than the injection of mice, and the in vitro product is very rich in antibody, whilst containing low amounts of contaminating proteins.

Antibodies, Monoclonal↗
PubMed1992

Non-random features of the repertoire expressed by the members of one V kappa gene family and of the V-J recombination.

The 5' and 3' flanking sequences of 14 members of the V kappa Ox (VK 4/5) gene family of BALB/c mice have been established. The family was unusual in the number of bases between the codon for Pro 95 and the heptamer sequence; most members contained four but there were also examples of none. A conserved leader sequence was used to amplify the genomic DNA of rearranged genes in order to analyze the spleen B cell repertoire of non-immunized animals. The library contained many members with virtually identical sequences to one or other of the already known members of the family. In addition, there were repeats of other sequences, allowing the definition of 12 hitherto undefined members of the family. Only 3 out of 96 could have originated by gene conversion, or as artefacts of the amplification procedure, and only 2 were putative somatic mutants. The frequency of expression of different members of the V kappa Ox gene family was not random, and some germ-line genes were unrepresented in the library. The high frequency of V kappa Ox1-J kappa 5 is in line with the dominance of this combination in the oxazolone response. An analysis of the junctional segment showed that although in most cases the diversity was due to trimming, there were exceptions indicating de novo additions (N or P bases). The average number of bases trimmed from the V kappa and the J kappa segments was not the same. There was no correlation in the number of bases trimmed from V kappa or J kappa in each recombination. The implications of asymmetric trimming in terms of the mechanism of recombination are discussed.

Animals↗
PubMed1991

Kinetic maturation of an immune response.

Is the affinity maturation of antibodies under thermodynamic or kinetic control, or both? We compared the physical constants of hapten binding by antibodies from 2-phenyl-5-oxazolone-specific hybridomas from primary, secondary and tertiary responses. In addition to an increase in equilibrium constant, there was a shift in the antibody repertoire after the primary response towards an immunoglobulin family with an extremely high on-rate constant. This shift occurred in spite of the average or below-average affinity of this group of antibodies. This is consistent with B-lymphocyte proliferation being subject to a kinetic selection, with a premium on binding target antigens rapidly, in parallel with a thermodynamic selection based on binding tightly.

Animals↗
PubMed1991

Mutation and selection during the secondary response to 2-phenyloxazolone.

The most characteristic feature of the mouse antibody response to the hapten 2-phenyloxazolone is the recurrent expression of the light-chain variable region Igk-VO chi 1 gene in its germ-line or mutated configuration. The analysis of somatic mutants of the Igk-VO chi 1 gene reported here indicates that, as found during the primary response, hypermutation is also activated during the secondary response. Somatic mutations in the Igk-VO chi 1 gene increased in sequences obtained at day 14 and day 21 in the primary response and again in the secondary response at days 3, 5, and 7. The ratio of replacement to silent mutations also increased, particularly between days 5 and 7, suggesting that a stage of negative selection operates on new somatic mutants generated in the secondary response. Most Igk-VO chi 1 mutants isolated in the secondary response had the features of selected memory clones (i.e., they carried mutations known to increase binding affinity for the hapten). However, some clones had chain-termination codons, and others had mutations predicting a nonfunctional light chain. At least three and possibly five of these clones also expressed the mutation characteristic of the memory response to 2-phenyloxazolone (His-34----Asn-34/Gln-34). We conclude that after a second antigenic challenge, new somatic variants, including some leading to the loss of antigen binding, are generated by hypermutation of cells derived from the memory pool.

Amino Acid Sequence↗
PubMed1991

Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51.

The microtubule-associated protein tau that is incorporated into paired helical filaments (PHFs) undergoes some form of aberrant posttranslational processing in Alzheimer disease. Difficulties in deciding which changes are critical for PHF formation stem in part from the lack of immunochemical markers specific for PHF tau. The only monoclonal antibody (mAb) that is known to react with PHF tau but not with the predominant normal adult tau species is mAb 423. Another mAb (7.51, described in this paper) recognizes a segment of tau that is included in the minimal recognition unit required by mAb 423. Unlike 423, which is PHF tau-specific, mAb 7.51 recognizes all PHF core-derived tau as well as native soluble tau and recombinant tau expressed in bacteria and so serves as a generic tau marker. Both epitopes are in the 12-kDa fragment released from the Pronase-resistant core of the PHF (which encompasses the tandem repeat region). The mAb 7.51 epitope requires segments located in the last two repeats, which are common to all tau isoforms. The mAb 423 epitope requires sequences located near both the N and the C terminus of the 12-kDa fragment common to three- and four-repeat tau isoforms. Fragments denatured by concentrated formic acid and SDS regain 423 reactivity when denaturing agents are removed. Since the primary amino acid sequences of PHF tau and normal tau are identical in the repeat region, we conclude that 423 reactivity also requires a modification(s) occurring within an approximately 90-residue segment that are not present in tau proteins so far described in the human brain.

Alzheimer Disease↗
PubMed1991

Colony assays for antibody fragments expressed in bacteria.

This paper describes procedures for the detection and selection of bacterial colonies expressing antibody fragments of desired antigen specificity. Fab and Fv fragments are detected in a filter assay in which bacterial colonies are grown on a master filter in contract with a second, antigen-coated filter. Ab fragments diffusing onto the second filter bind antigen directly and specifically and are detected with a monoclonal antibody directed against a myc-tag sequence fused to the carboxy-terminal end of the light chain or heavy chain (direct assay). Single-chain Fv (scFv) in which the VH and V1 sequences are joined by a short linker peptide are detected by a modified procedure in which scFv are immobilized on filters coated with the anti-myc-tag sequence and subsequently detected by specific binding to radiolabeled antigen (indirect assay). A single positive bacterial colony expressing antigen-specific Fv (or scFv) can be recovered among at least 10,000 negative colonies using the procedures described. The direct assay has been successfully used to discriminate Fv fragments which express point mutations known to increase the binding affinity of antibodies to the hapten 2-phenyl-oxazolone. The procedures described may thus prove generally useful for the selection of antigen-specific clones expressed in bacteria and/or higher-affinity variants of such antibodies.

Antibodies↗
PubMed1991

Man-made antibodies.

Monoclonal antibodies can now be genetically engineered and endowed with new properties. In the future, gene technology could enable antigen-binding fragments to be made by exploiting repertoires of variable domain genes derived from immunized animals and expressed in bacteria. How readily can this approach be extended to production of 'in vitro' repertoires of variable domain genes, and obviate the immunization of animals?

Animals↗
PubMed1991

The identification of the beta 2-microglobulin binding antigen encoded by the human CD1D gene.

Human cluster of differentiation (CD1) is a family of cell surface glycoproteins composed of a 43-49-kDa heavy chain non-covalently associated with beta 2-microglobulin. Five human CD1 genes have been detected and cloned. Three genes (CD1A, -B and -C) encode the serologically defined CD1a, -b and -c antigens. Thus two genes remain, CD1D and CD1E, whose protein products have not been characterized so far. This report describes how a beta-galactosidase-CD1D fusion protein was used to raise specific antisera and a monoclonal antibody against the CD1D gene product. The monoclonal antibody defines a cell surface molecule expressed on a cortical thymocyte cell line and is composed of a 49-kDa heavy chain associated with beta 2-microglobulin, which is serologically distinct from CD1a.

Antibodies, Monoclonal↗