Fcgamma receptor 1 (CD64), a target beyond cancer.

Immunotoxins are powerful tools to specifically eliminate deviated cells. Due to the side effects of the original immunotoxins, they were only considered for the treatment of cancer as in these cases, the potential favourable effect outweighed the unwanted toxic side effects. Over time, many improvements in the construction of immunotoxins have been implemented that circumvent, or at least strongly diminish, the side effects. In consequence this opens the way to employ these immunotoxins for the treatment of non-life threatening diseases. One such category of disease could be the many chronic inflammatory disorders in which an uncontrolled interaction between inflammatory cells leads to chronicity. In several of these chronic conditions, activated macrophages, which are characterised by an increased expression of CD64, are known to play a key role. In this review we discuss the data presently available on elimination of activated macrophages through CD64 immunotoxins in several animal models for chronic disease. A chemically linked complete antibody with the plant toxin Ricin-A, proved very effective and provided proof of concept. Subsequently, the development towards genetically engineered, fully human, multivalent single chain based immunotoxins that have diminished immunogenicity, is discussed. The data show that the specific elimination of activated macrophages through CD64 is indeed beneficial for the course of disease. As opposed to other methods used to inactivate or eliminate macrophages, with the CD64 based immunotoxins only the activated population is killed. This may open the way to apply these immunotoxins as therapeutics in chronic inflammatory disease.

New DNA markers with increased informativeness show diminished support for a chromosome 5q11-13 schizophrenia susceptibility locus and exclude linkage in two new cohorts of British and Icelandic families.

Genetic linkage of schizophrenia to markers at 5q11.2-13.3 had been reported previously in five Icelandic and two British families, but attempts at replication in independent samples have been unsuccessful. We report here an update on the diagnoses and results of linkage analyses using newer highly polymorphic microsatellite markers at or near the loci D5S76 and D5S39 in the original sample of pedigrees and in two new family samples from Iceland and from Britain. The new results show a reduction in evidence for linkage in the original sample and evidence against linkage in the two new family samples. Although it is possible that a rare locus is present, perhaps in the region 5p14.1-13.1 rather than 5q11.2-13.3, it appears most likely that the original positive lod scores represent an exaggeration of the 'true' lod scores due to random effects and that the small lod scores we now obtain could have arisen by chance.

Molecular and cytogenetic investigations of the fragile X region including the Frax A and Frax E CGG trinucleotide repeat sequences in families multiplex for autism and related phenotypes.

We undertook molecular and cytogenetic analyses in 25 families multiplex for autism and related disorders. Three of the multiplex families exhibited fragile X, and the affected offspring all exhibited CGG triplet repeat insertion mutations in the FMR-1 gene. One of these families contained an affected pair of monozygotic female twins. Both had similar-sized CGG triplet repeat expansions, but different phenotypic manifestations. One suffered from autism and the other from mild mental retardation and marked social anxiety. PCR and Southern hybridization analysis of the CGG repeat sequences characterizing fragile X A (Frax A) and E and the methylation status of FMR-1 showed no evidence of abnormal CGG repeat expansion or FMR-1 hypermethylation in the remaining 22 multiplex families. Moreover, there was no correlation between the Frax A or E (CGG)n repeat length with affected status, nor any association with the low-level (< 3 %) expression of cytogenetic fragility at Xq27 previously reported in these families. Our findings indicate that most instances of recurrence in families multiplex for autism and related disorders are not accounted for by Frax A and E. They also indicate that the phenotypic manifestations of Frax A may be influenced by stochastic, environmental and other biological factors.

Hybridization of the variable number of tandem repeat core oligonucleotide GNNGTGGG to 350 cosmids from chromosome 7.

An efficient method using short oligonucleotides is described for the isolation of minisatellite markers of the type variable number of tandem repeats (VNTR). It is used to screen 350 cosmids from chromosome 7 for the presence of such potentially polymorphic DNA segments. From the number of hybridization signals to chromosome 7 cosmids, I estimate the human genome to have at least about 15000 VNTR loci containing minisatellite core sequences, which is well above previous estimates.

Oligonucleotides as short as 7-mers can be used for PCR amplification.

Amplification of DNA sequences using the polymerase chain reaction (PCR) requires as primers two oligonucleotides, which are carefully designed for length and G/C content. Such primers are generally between 18 and 30 bases long so that the primer sequences can amplify a unique sequence in the target genome; they should possess a minimal degree of secondary structure. We have tested the minimum length of G/C-rich and palindromic oligonucleotides to be used as primers in PCR. Oligonucleotides with sequences corresponding to the recognition sites of rare restriction enzymes were used on the DNA of vector constructs as model template DNA. Surprisingly, we found specific amplification with a low background over a wide range of temperatures for oligonucleotides as short as 7 nucleotides. This findings contradicts the previously reported empirical relationship between oligonucleotide length and ability to trigger amplification and points to the complex relationship between thermodynamic and kinetic criteria in relation to PCR. This technique should lead to new application in the cloning and screening of complex genomes.

Screening cosmid libraries with oligonucleotides corresponding to splice-site consensus sequences.

To facilitate the identification of genes within genomic DNA, we have developed a method based on the use of short oligonucleotides designed from the consensus sequences of splice sites. We describe here the hybridization and washing conditions under which such oligonucleotides can be used to screen cosmid libraries. We confirm the presence of genes within cosmids identified by screening with one oligonucleotide by showing that DNA isolated from such cosmids will hybridize to another splice-site oligonucleotide.

Characterization of a human chromosome 4 flow-sorted cosmid library.

A cosmid library has been constructed from a hamster x human hybrid cell line and gridded into 270 microtiter plates containing a total of 25,920 single colonies. Approximately 84% of the recombinants contain human material, with an average length of 29 kb. This library represents a nearly three-fold coverage of human chromosome 4. We investigated this library for presumptive genes, using a set of oligonucleotides detecting SpI and splice-site consensus sequences. The presence of simple repeat motifs was investigated in the cosmids using the oligonucleotides (GGATTT)3, (GGAT)4, (CAC)5, (GCC)5, (AGC)5, (GATA)4, (GACA)4, and (CA)8 as hybridization probes.

Microsatellite polymorphisms for chromosome 5 bands q11.2-q13.3.

The genes for spinal muscular atrophy (SMA) and a possible subtype of schizophrenia (SCZD1) have been mapped to chromosome 5q11.2-q13.3. DNA markers have been mapped to 5q11.2-q13.3 using a hybrid cell line deleted for this region [Gilliam et al., Genomics 1989;5:940-944]. Genomic lambda clones for these markers facilitated the identification of highly polymorphic microsatellites. A total of ten microsatellites were identified and sequenced. Of these, seven were found to be polymorphic. Four had polymorphism information content values > 0.7. New polymorphic microsatellites were sequenced for D5S76, D5S125, D5S39, D5S127 and HEX-B. Two-point and multipoint analysis in non-CEPH pedigrees confirmed that the microsatellites were in tight linkage with each other. These new microsatellites will increase the efficiency of linkage analysis for these disorders.

Leukaemic T cells from patients with chronic lymphocytic leukaemia of T-cell origin respond to Staphylococcus aureus enterotoxin superantigens.

We investigated the in vitro responsiveness of peripheral blood lymphocytes from two patients with T-cell chronic lymphocytic leukaemia (T-CLL) to Staphylococcus aureus enterotoxin (SE) superantigens. T-cell receptor (TcR) alpha beta (V beta 7.1)-expressing CD4+ leukaemic T cells from patient HE (white blood cell count 480,000/microliters) proliferated in response to SEA and, only at 1000-fold higher concentrations, to SEB, SED, and SEE. CD4+CD8+ TcR alpha beta (V beta 12.1)-expressing leukaemic T cells from patient KO (white blood cell count 120,000/microliters) were activated by SEB but not by the other tested SEs. In both instances, the activation of leukaemic T cells by SE was dependent on the presence of HLA-DR+ cells. Southern blot analysis of TcR beta gene rearrangement confirmed that the proliferating cells were derived from the leukaemic T-cell clone and not from contaminating normal T cells. These data indicate that leukaemic T cells from patients with T-CLL exert a clonally variable responsiveness to SE superantigens. We conclude that recognition of specific antigen and subsequent signal transduction can be initiated via the TcR of leukaemic T-CLL cells.

On the essence of "meaningless" simple repetitive DNA in eukaryote genomes.

Various kinds of simple tandemly repetitive DNA sequences are abundantly interspersed in the genomes of practically all eukaryotic species studied. The comparatively elevated mutation rates of simple repeat blocks result in highly polymorphic and therefore extremely informative investigation systems for studies on forensic, ecological and genetic relationship questions. Recently the techniques for analyzing simple repeats have achieved great effectivity and simplicity. Beyond their utility as tools for differentiation and individualization, certain of these repeated elements harbor quite unexpected qualities which may be discussed in the context of their biological meaning. i) A specific subset of simple (cac)n or (gtg)n repeats is expressed in mature mRNA and total cellular RNA. ii) Despite the apparently high mutation rate certain (gt)n or mixed (gt)n/(ga)m stretches of intronic simple repeats are preserved in immunologically relevant genes for at least 70 x 10(6) years and they bind nuclear protein molecules with high affinities. Consequently in addition to their tool character, the biological aspects of simple repeated DNA should be taken into consideration.

On the potential of simple repetitive DNA for fingerprinting in clinical, forensic, and evolutionary dynamic studies.

The purpose of this review is to discuss critically the practical meaning of a specific genome component, simple repetitive desoxyribonucleic acid (DNA) sequences as clinical and forensic and diagnostic and research tools. Previously, multilocus DNA fingerprinting was the major technology employed to visualize such simple repeat sequences. This technique enables many polymorphic loci to be simultaneously detected thus yielding vast amounts of information. With the advent of enzymatic DNA amplification via the polymerase chain reaction (PCR), individual simple repeat loci can be demonstrated, theoretically even from single DNA molecules and so a wealth of additional approaches have also become feasible. In general investigating, small, known, single copy parts of genomes have not posed truly insurmountable problems if enough material was available. There have even been a few (anecdotal) reports on the amplification of simple repeats from ancient DNA (see, e.g. [30]. Here we would like to after a solid basis for an earnest discussion of the applications of these simple repetitive sequences using various methodological approaches relevant for clinical diagnosis, setting aside the obvious unsolved mysteries of their biology.

Identification of genes using oligonucleotides corresponding to splice site consensus sequences.

The identification of genes in genomic DNA presents challenging technical difficulties. We show here the feasibility of using short oligonucleotides based on the consensus sequences surrounding intron-exon junctions to detect random phage and cosmid clones containing genes both through the analysis of DNA blots and by direct screening. Three degenerate oligonucleotides, a 10-mer corresponding to the 5' splice junction and a 9-mer and a 15-mer corresponding to the 3' splice junction, were tested on the known intron-exon boundaries of the cloned human proteolipid protein (PLP) gene at hybridization and washing temperatures appropriate to their length and composition. All predicted hybridizations were observed. The oligonucleotides were also used to identify random genomic plasmid and cosmid clones containing putative intron-exon junctions; the presence of genes in these clones was supported by RNA blot analysis and by cross-hybridization to DNA from other species. This technique should facilitate the identification of genes for inherited diseases by positional cloning studies and will assist in the identification of genes in random clones for the human genome project.

Simple repeats are not found more abundantly at G/C-rich regions.

Three hundred and fifty cosmids from chromosome 7, previously analyzed for the presence of rare-cutting restriction enzyme sites, were analyzed for the presence of microsatellite sequences [(CA)n or (CT)n repeats]. Of these, 147 cosmids were found to contain at least one (CA)n repeat unit and 51 cosmids contained at least one (CT)n repeat unit. No evidence was found for the prevalence of microsatellite repeat units in the vicinity of rare-cutter restriction enzyme sites.

PCR amplification products are of limited use for the study of DNA/protein interaction.

Conventional methods for labeling double-stranded DNA lead to high specific activity. Yet they often alter the target DNA sequence to such an extent as to prevent a meaningful protein/DNA interaction analysis. Therefore we tried to establish a polymerase chain reaction (PCR)-based method which allows radiolabeling to high specific activity and should maintain the protein binding capability of small double stranded DNA fragments. By using PCR it is possible to label double stranded DNA to high specificity, but the protein binding capability of such DNA is drastically reduced.

Linkage disequilibrium between two highly polymorphic microsatellites.

The PCR was used to amplify genomic DNA from two microsatellite (dC-dA)n.(dG-dT)n sequences found to be present in the same chromosome 5 genomic clone. Analysis of the haplotype frequencies of these two interspersed repeat sequences in individuals showed strong allelic association or linkage disequilibrium. Six alleles were found for p599 (CA)n with a PIC value of 0.71 and 8 alleles were seen for lambda 599 (CA)n with a PIC value of 0.74. The two microsatellites are separated by approximately 7 kb. Analysis of the length variations for the two microsatellites showed that they were positively correlated, a finding that has no obvious explanation. The strong linkage disequilibrium found demonstrates stability during evolution for these novel markers. Therefore they should be powerful new tools for studying genetic drift and admixture of populations. Furthermore, disequilibrium data from microsatellites can be used in the fine mapping and cloning of disease genes.

A cosmid clone for the 5HT1A receptor (HTR1A) reveals a TaqI RFLP that shows tight linkage to dna loci D5S6, D5S39, and D5S76.

A human neuroreceptor clone (G21), which was isolated by cross-hybridization with the human clone for the beta 2-adrenergic receptor, has recently been shown to encode the gene for the 5HT1A receptor (HTR1A) subtype. In situ hybridization to human metaphase chromosomes mapped the G21 sequence to chromosome 5 at bands 5q11.2-q13. The clone G21 recognizes a SacI RFLP with low heterozygosity (0.13). To increase the informativeness of the HTR1A locus we have isolated two new cosmid clones containing the receptor gene. No polymorphic microsatellites were present in the cosmids. However, one cosmid revealed a new TaqI RFLP that showed tight linkage to new highly polymorphic microsatellites for the loci D5S76, D5S39, and D5S6 in seven British and Icelandic reference pedigrees (maximum LOD of 13.2 with D5S76).