Biomedical subjects

J Hampe

Publications and source records attributed to J Hampe.

16 recordsLinked to original sources

PubMed1999

Tumour necrosis factor alpha and interleukin 1beta in relapse of Crohn's disease.

BACKGROUND: Concentrations of proinflammatory cytokines are increased in the intestinal mucosa of patients with active Crohn's disease. Experimental immunotherapeutic interventions with anticytokine agents in refractory Crohn's disease show that tumour necrosis factor alpha (TNF alpha) may be an important mediator of inflammation. We investigated the relation between production of TNF alpha and interleukin 1beta by mononuclear cells of the colonic lamina propria in patients with remitting Crohn's disease and the risk of relapse. METHODS: We followed up 137 patients with Crohn's disease in steroid-induced remission for 1 year. Secretion of proinflammatory cytokines (tumour necrosis factor alpha [TNF alpha] and interleukin 1beta) was assessed after short-term culture of human lamina propria mononuclear cells. FINDINGS: Increased secretion of TNF alpha and interleukin 1beta were predictive for acute relapses within the next year. Site and extent of disease, baseline demographics, and serum acute-phase proteins had little predictive value. INTERPRETATION: TNF alpha is important as a target molecule for immune interventions in Crohn's disease. The capacity to produce TNF alpha or interleukin 1beta may identify patients who would benefit from anti-inflammatory remission maintenance.

Biomarkers↗

PubMed1999

A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort.

Inflammatory bowel disease (IBD) is characterized by a chronic relapsing intestinal inflammation, typically starting in early adulthood. IBD is subdivided into two subtypes, on the basis of clinical and histologic features: Crohn disease and ulcerative colitis (UC). Previous genomewide searches identified regions harboring susceptibility loci on chromosomes 1, 3, 4, 7, 12, and 16. To expand our understanding of the genetic risk profile, we performed a 9-cM genomewide search for susceptibility loci in 268 families containing 353 affected sibling pairs. Previous linkages on chromosomes 12 and 16 were replicated, and the chromosome 4 linkage was extended in this sample. New suggestive evidence for autosomal linkages was observed on chromosomes 1, 6, 10, and 22, with LOD scores of 2.08, 2.07, 2.30, and 1.52, respectively. A maximum LOD score of 1.76 was observed on the X chromosome, for UC, which is consistent with the clinical association of IBD with Ullrich-Turner syndrome. The linkage finding on chromosome 6p is of interest, given the possible contribution of human leukocyte antigen and tumor necrosis-factor genes in IBD. This genomewide linkage scan, done with a large family cohort, has confirmed three previous IBD linkages and has provided evidence for five additional regions that may harbor IBD predisposition genes.

Cohort Studies↗

PubMed1998

Genomic difference analysis by two-dimensional DNA fingerprinting reveals typical changes in human low-grade gliomas.

Cytogenetic and molecular analyses such as allelotyping studies have revealed several genetic changes typical for human glial neoplasms. However, most studies to date have involved malignant gliomas and thus are likely to reflect late events of tumor progression. To elucidate the initial events of glial tumor growth, we performed a genome-wide search for genetic alterations in the DNA of 43 low-grade gliomas as compared to the constitutional DNA of the patients' peripheral blood leucocytes using the two-dimensional (2D) DNA fingerprint approach. Reliable results were obtained for 28 blood/tumor sample pairs (13 astrocytomas, 9 pilocytic astrocytomas, 1 oligodendroglioma, 3 oligoastrocytomas, and 2 ependymomas). DNA was digested with the restriction enzyme HaeIII and the resulting fragments were separated on 2D gels according to size and sequence in the first and second dimensions, respectively. Patterns of hundreds of spots were generated by hybridization with four different mini- and microsatellite core probes. A total of 655 to 1,122 spots could be visualized per sample. Comparison of blood and tumor spot patterns revealed two to 11 reproducible changes per patient. Most of the differences were spot losses (77.1%), while the others appeared to be gains or amplifications. Exactly the same changes were found in tumor recurrences which lacked histological signs of progression. When comparing different patients, many of the affected spots tended to cluster in particular areas of the gel as revealed by computer-aided comparison of all spot patterns. Eleven different spot clusters were identified which may correspond to several major deletion targets. This study provides the basis for the future molecular cloning of the candidate tumor suppressor genes affected by the common spot losses and will allow new insights into the genetic mechanisms of glial tumorigenesis.

Adolescent↗

PubMed1998

The interferon-gamma gene as a positional and functional candidate gene for inflammatory bowel disease.

Epidemiological and genome-wide linkage analyses have provided firm evidence for a genetic component in the pathogenesis of inflammatory bowel disease. The linkage regions on chromosomes 12 and 16 have been replicated in several independent samples. These represent the best positional evidence in the search for inflammatory bowel disease susceptibility genes. While systematic association and physical mapping studies in these regions are under way, the direct analysis of immunologically relevant genes as positional and functional candidates may provide a shortcut in this process. The interferon-gamma gene resides in the chromosome 12 linkage region near the marker D12S83. Interferon-gamma is an important proinflammatory cytokine in the interleukin-12 cascade and has been implicated in the pathogenesis of mucosal inflammation. We tested this gene for evidence of linkage and association in 133 German multiplex families and 506 single patients with their parents. An intragenic, highly informative CA-repeat marker in intron 1 of the gene was typed using fluorescence-labeled polymerase chain reaction and analysis on an automated sequencer. In the nonparametric linkage analysis using GENEHUNTER, a nonsignificant maximum LOD score of 0.67 was obtained. The transmission disequilibrium test for association was negative (P > or = 0.22) for Crohn's disease, ulcerative colitis, and the combined inflammatory bowel disease phenotype. In summary, the findings make interferon-gamma a very unlikely candidate for the major susceptibility gene in the chromosome 12 linkage interval. Future efforts can concentrate on other transcripts in the region.

Base Sequence↗

PubMed1998

Genetic analysis of inflammatory bowel disease in a large European cohort supports linkage to chromosomes 12 and 16.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is a complex disorder of unknown etiology. Epidemiological investigations suggest a genetic basis for IBD. Recent genetic studies have identified several IBD linkages. The significance of these linkages will be determined by studies in large patient collections. The aim of this study was to replicate IBD linkages on chromosomes 12 and 16 in a large European cohort. METHODS: Three hundred fifty-nine affected sibling pairs from 274 kindreds were genotyped using microsatellite markers spanning chromosomes 12 and 16. Affection status of the sibling pairs was defined as Crohn's disease (CD) or ulcerative colitis (UC). RESULTS: Nonparametric statistical analyses showed linkage for both chromosomes. Two-point results for chromosome 12 peaked at D12S303 (logarithm of odds [LOD], 2.15; P = 0.003) for CD and at D12S75 (LOD, 0.92; P = 0.03) for UC. Multipoint analyses produced a peak LOD of 1.8 for CD. Chromosome 16 showed linkage for CD at marker D16S415 (LOD, 1.52; P = 0.007). Multipoint support peaked above markers D16S409 and D16S411 (LOD, 1.7). CONCLUSIONS: These data are consistent with linkage of IBD to chromosomes 12 and 16. The replication of genetic risk loci in a large independent family collection indicates important and common susceptibility genes in these regions and will facilitate identification of genes involved in IBD.

Chromosomes, Human, Pair 12↗

PubMed1998

POPSIM: a general population simulation program.

MOTIVATION: The investigation of common disorders of polygenic inheritance using both population-based association designs and non-parametric linkage analysis within families is gaining increasing importance. We have created a program that allows for the flexible simulation of populations as a tool to investigate the properties of population-based mapping approaches. RESULTS: We have created a population simulation program, POPSIM, that (i) creates a virtual representation of every individual, (ii) makes no prior assumptions but the Mendelian rules and (iii) allows populations of several million individuals in size to be generated and to be followed over hundreds of generations. The parameters of the disease model, population structure and population expansion rate can be specified. Flexible sampling options exist that allow samples of families and individuals to be drawn at any given point during the population history. The program may be a useful tool in the study of the influence of genetic drift, recombination and admixture on the generation and maintenance of linkage disequilibrium in populations, as well as the evaluation of stochastic sampling characteristics of families and individuals conditional on a complex genetic phenotype from homogeneous and heterogeneous populations. AVAILABILITY: The source code as well as Sun and Windows NT4.0 console executables of the program are available under http://www.ukrv. de/ch/medgen/html/benutzer/j.hampe/popsim. html. CONTACT: J. Hampe@mucosa.de

Computational Biology↗

PubMed1998

Activation of nuclear factor kappa B inflammatory bowel disease.

BACKGROUND: Expression of pro-inflammatory cytokines is increased in the intestinal lamina propria of patients with inflammatory bowel disease (IBD). Nuclear factor kappa B (NF kappa B) controls transcription of inflammation genes. On activation, NF kappa B is rapidly released from its cytoplasmic inhibitor (I kappa B), transmigrates into the nucleus, and binds to DNA response elements in gene promoter regions. AIMS: To investigate whether increased activation of NF kappa B is important in IBD and may be down-regulated by anti-inflammatory treatment. METHODS: Activation of NF kappa B was determined by western blot assessment and electrophoretic mobility shift assay in nuclear extracts of colonic biopsy samples as well as lamina propria mononuclear cells. RESULTS: Nuclear levels of NF kappa B p65 are increased in lamina propria biopsy specimens from patients with Crohn's disease in comparison with patients with ulcerative colitis and controls. Increased activation of NF kappa B was detected in lamina propria mononuclear cells from patients with active IBD. Corticosteroids strongly inhibit intestinal NF kappa B activation in IBD in vivo and in vitro by stabilising the cytosolic inhibitor I kappa B alpha against activation induced degradation. CONCLUSIONS: In both IBDs, but particularly Crohn's disease, increased activation of NF kappa B may be involved in the regulation of the inflammatory response. Inhibition of NF kappa B activation may represent a mechanism by which steroids exert an anti-inflammatory effect in IBD.

Adolescent↗

PubMed1997

'Complicated' autosomal dominant familial spastic paraplegia is genetically distinct from 'pure' forms.

BACKGROUND: The familial spastic paraplegias (FSPs) are hereditary neurodegenerative disorders with an unknown pathogenesis. Pure and complicated forms are currently differentiated on clinical grounds. To date, no linkage studies in complicated FSP have been reported, and candidate genes have not been suggested. Three different gene loci responsible for pure autosomal dominant FSP and 1 for pure autosomal recessive FSP recently have been found. This raises the question of whether the complicated forms may also be linked to any of these loci. OBJECTIVE: To investigate whether complicated autosomal dominant FSP is allelic to any of the pure forms with defined loci. DESIGN: Clinical characterization of a large kindred that included 4 generations and multipoint linkage analyses. SETTING: Universitätsklinikum Charité, Humboldt-Universität Berlin, Neurologische Klinik und Poliklinik, Berlin, Germany. PATIENTS: Twenty-six family members, 13 of whom were affected. RESULTS: Thirteen members of a large family of 4 generations experienced a slowly progressive syndrome of spastic paraplegia. Hypomimia, bradykinesia, axial and limb rigidity, supranuclear gaze palsy, dysarthria, bladder and sphincter disturbances, cerebellar signs, and epilepsy were noted as additional features in some of the affected individuals. The mean age at onset was 20 years (range, 5-30 years), and the pattern of transmission was compatible with an autosomal dominant mode of inheritance. The CAG-repeat expansions in the spinocerebellar ataxia type 1 and Machado-Joseph disease genes were not found. Linkage analysis with the use of a panel of (AC)n dinucleotide repeat markers from the Généthon map demonstrated exclusion of all 4 FSP loci recently mapped by linkage to pure forms of FSP on chromosomes 14q, 2p, 15q, and 8. CONCLUSIONS: Complicated FSP in this family is not linked to any of the known pure FSP loci, including the recessive one. Therefore, the clinical differentiation of both forms still is of major relevance.ACKG

Aged↗

PubMed1997

Cloning of minisatellite-containing sequences from two-dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low-grade gliomas of different patients.

Two-dimensional (2-D) DNA fingerprinting was used to investigate genomic changes in human low-grade gliomas of different subtypes. DNA variations were identified in the 2-D hybridization patterns as spot losses or gains. Computer-aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2-D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)-amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus-specific probe for the rehybridization of the 2-D blots. One of these probes detected a spot loss in 7 of 28 low-grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus-specific probes that may be instrumental in the discovery of the critical early events of glioma pathogenesis.

Chromosome Mapping↗

PubMed1997

A novel standardization method for two-dimensional DNA fingerprints.

Two-dimensional (2-D) DNA fingerprinting is a technique that allows for parallel genome analysis through the simultaneous detection of up to 500 mini- or microsatellite loci on a 2-D gel. Separation is performed according to size and melting temperature in the gel. In the application of this technique in genome analysis, a standardized method for the identification of individual spots is required. However, due to the polymorphic nature of up to 80% of the spots, existing standardization methods that have been primarily developed for 2-D protein patterns are not suitable for this task. We developed a robust method that standardizes 2-D DNA fingerprint spots on the basis of melting temperature - or denaturing gradient position - and fragment size. An external marker was used as a basis for standardization. A normalization surface was calculated over the gel dimensions by adapting an established numerical iteration technique previously used in physics termed "relaxation method". The relaxation method works robustly with the irregularly spaced marker spots. The evaluation of the method for a spot of preknown position derived from the TP53 gene revealed a median observed error below 1% for fragment length and denaturing gradient position. The search for candidate minisatellite loci in genomic difference analysis depends on the reliable identification of alleles of this locus in different individuals. We proved experimentally that alleles of a single minisatellite locus cloned from a 2-D gel cluster on an isothermal line can be reliably identified using the presented standardization method. In conclusion, a standardization tool for a broader application of 2-D DNA fingerprinting in both tumor analysis and possibly parallel mutation screening is now available.

Alleles↗

PubMed1997

The gene for autosomal dominant craniometaphyseal dysplasia maps to chromosome 5p and is distinct from the growth hormone-receptor gene.

Craniometaphyseal dysplasia (CMD) is an osteochondrodysplasia of unknown etiology characterized by hyperostosis and sclerosis of the craniofacial bones associated with abnormal modeling of the metaphyses. Sclerosis of the skull may lead to asymmetry of the mandible, as well as to cranial nerve compression, that finally may result in hearing loss and facial palsy. We have analyzed a large German kindred with autosomal dominant (AD) CMD and found tight linkage between the disorder and microsatellite markers on chromosome 5p (maximum two-point LOD score 4.82; theta = 0). Our results clearly establish the existence of a locus for AD CMD on central chromosome 5p (5p15.2-p14.1). This region overlaps with the mapping interval of the growth hormone-receptor (GHR) gene (5p14-p12), which is known to be involved in the mitogenic activation of osteoblasts. Therefore, we tested the GHR gene as a candidate gene. However, recombination events between the CMD locus and the GHR gene identified in two members of this family clearly exclude this candidate.

Chromosome Mapping↗

PubMed1996

Marker pattern instabilities as a major cause of reproducibility problems in two-dimensional DNA fingerprinting.

Two-dimensional (2-D) DNA fingerprinting is a promising technique for multilocus analysis of eukaryotic genomes. It has been successfully applied to the detection of DNA variation in tumors, to linkage analyses and to genomic comparisons of inbred mouse strains. However, there are still problems with inter-gel comparisons of 2-D DNA typing patterns as documented by the inter-gel reproducibility rates reported in the literature, which range from 84 to 98%. The basis for standardization in almost all of these studies has been a set of lambda fragments (digested separately with the restriction enzymes HaeIII, RsaI, Bg/I) that produces a spot pattern scattered across the gel. These spots are used as markers for gel comparisons. Since we noticed considerable variations in the marker spot patterns, we evaluated the properties of the lambda marker using both computer simulation and an empirical analysis of forty independent consecutive gels from our laboratory. We explain the instabilities of the spot pattern on the basis of the melting properties of the individual lambda fragments. A subset of spots is presented that has been stable in all our experiments. Only this set of spots should be used for gel standardization purposes until a new, completely reproducible marker becomes available. Finally, suggestions for an improved marker system are made.

Bacteriophage lambda↗

PubMed1995

Parallel genome analysis by one- and two-dimensional DNA fingerprinting in human gliomas.

The detection of DNA variation in cancers is an important step in elucidating the mechanism of tumorigenesis. Using the strategy of multipoint genome analysis we detected many differences between glioma-derived and constitutional DNA by customary DNA fingerprinting with simple repetitive oligonucleotide probes. Amplification of the epidermal growth factor receptor (EGFR) gene has been found to be easily detectable as new or highly intensified bands in one-dimensional (1-D) DNA fingerprints of glioblastoma DNA generated with probes (GTG)5 or (GT)8. However, in most low-grade astrocytomas, 1-D DNA fingerprinting has failed to reveal any genomic abnormalities. In these cases a two-dimensional (2-D) technique was successfully employed that is based on size separation in neutral gels followed by sequence-dependent separation in denaturing gradient gels and hybridization with several mini- and microsatellite core probes. The hundreds of spots visualized with this technique were used to detect subtle changes probably occurring as the initial steps of tumorigenesis in human gliomas. On average, five of the approximately 580 spots generated by probes CAC and 33.6 were found to be altered in tumor DNA; 80% of the alterations were spot losses, the rest being spot gains or amplifications. Computer-based image analysis using an external lambda marker provided a stringent way to compare spot patterns generated by 2-D DNA fingerprinting. In comparisons performed between typing patterns generated on the same gel, 99% of truly identical spots were confirmed by the software. In intergel comparisons 84% of identical spots were matched on the basis of the marker information alone.

Base Sequence↗

PubMed1993

Oligonucleotide DNA fingerprinting: results of a multi-center study on reliability and validity.

We report the results of an empirical study of 256 paternity cases referred to 7 different German laboratories for DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5. All parameters characteristic of such multilocus DNA fingerprints were found to differ significantly between the contributing centres. Despite these differences, clear-cut decisions between paternity and non-paternity could be made in all but one case. Furthermore, we found no systematic deviation of the gel-phenotype distribution among trios from random expectation as derived from commonly adopted analytical models. Thus, we conclude that oligonucleotide DNA fingerprinting is a robust and reliable means for the resolution of paternity cases.

Base Sequence↗

PubMed1993

Paternity testing with oligonucleotide multilocus probe (CAC)5/(GTG)5: a multicenter study.

The statistical analysis is reported of 256 paternity cases referred to seven different German laboratories for multilocus DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5 and restriction enzyme HinfI. All parameters characteristic of multilocus DNA fingerprints were found to differ significantly between the contributing centres: the number of analyzed gel positions, the number of bands scored per individual, the probability of occurrence of a band at a particular position, and the band-sharing probabilities between the mother and both child and alleged father. Despite these differences, paternity cases could be divided clearly into two distinct subgroups on the basis of (i) offspring bands that could not be assigned to either the mother or the alleged father and (ii) the extent of band-sharing between child and alleged father. This partitioning, which is likely to correspond to true and false paternity, confirms previous findings for other multilocus probes. A goodness-of-fit test on the normalized number of bands scored per individual revealed no systematic deviations from commonly adopted analytical models regarding electrophoretic bands as independent entities. Log10-likelihood ratios of paternity vs. non-paternity were calculated utilizing one of these models, and a clear-cut partitioning was again obtained which coincides with that mentioned before. Only one case could not be decided unambiguously, and was either due to two independent mutations or to a close relative of the alleged father being the true father.

Child↗

PubMed1992

Oligonucleotide fingerprinting as a means to identify and survey long-term cultured B cell hybridomas and T cell lines.

Common problems encountered during cell culture are cross-contamination, instability, and inadvertent exchange of cells. Here we report on the application of oligonucleotide fingerprinting as a simple and efficient method to screen hybridomas and T cell lines. Among the fingerprint probes tested, the simple repetitive oligonucleotide (CAC)5/(GTG)5 proved to be most useful for obtaining many fragments specific for each cell line. Because of variable loss of chromosomes, cloned hybridoma cells from one fusion exhibit different fingerprint patterns. Thus, antibody-secreting B cell hybridomas can be distinguished easily even when they originate from the same fusion. Furthermore, we were able to monitor the genomic integrity of myelomas and hybridomas over a period of more than 2 years, thereby highlighting long-term stability. Another application of the method is the control of T cell lines requiring irradiated or mitomycin-treated feeder cells for continuous growth or cloning. There cell lines are always threatened to be overgrown by feeder cells that have escaped from the lethal pretreatment. T cell clones of one individual, known to display differently rearranged T cell receptors, did not show differences in their fingerprint patterns. However, in EBV-transformed cloned B cells, slight differences between clones from the same donor were identified.

B-Lymphocytes↗