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Biomedical subjects

C Milstein

Publications and source records attributed to C Milstein.

At least 55 records · Page 3Linked to original sources
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

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.

Aging↗
PubMed1994

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.

Computer Systems↗
PubMed1993

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.

Amino Acid Sequence↗
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↗