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Biomedical subjects

U Lehmann

Publications and source records attributed to U Lehmann.

At least 37 records · Page 2Linked to original sources
PubMed2001

Amplification of growth regulatory genes in intraductal breast cancer is associated with higher nuclear grade but not with the progression to invasiveness.

Ductal carcinoma in situ (DCIS), as an identifiable progenitor lesion of invasive breast cancer, represents a morphologically, biologically, and prognostically heterogeneous disease. It is not clear which molecular mechanisms are involved in progression to infiltrative growth. In this study, 83 DCIS classified according to the Van Nuys grading scheme were examined for amplification of growth regulatory genes that have been found to be amplified in invasive breast cancer (c-erbB2, topoisomerase IIalpha, c-myc, and cyclinD1 genes). Exact quantification of gene amplification was enabled by a combination of laser microdissection of paraffin-embedded tissue with real-time PCR. In DCIS, gene amplifications of all tested genes were found. The most frequently amplified gene was c-erbB2 found in 21 of 83 (25%) cases. Amplification of the other genes under investigation was observed in 4% to 6% of cases, high-grade DCIS being predominantly affected. High-grade DCIS differed significantly from low- and intermediate-grade DCIS in frequency and level of c-erbB2 amplification. In addition, high-grade DCIS revealed an accumulation of genetic aberrations. Amplification status in pure in situ lesions did not differ from intraductal carcinoma with an infiltrative component, indicating that although associated with a higher nuclear grade gene amplification might not represent an independent prognostic marker of disease progression.

Breast Neoplasms↗
PubMed2001

Heterotopic ossifications in patients after severe blunt trauma with and without head trauma: incidence and patterns of distribution.

OBJECTIVE: To investigate the incidence and distribution of heterotopic ossifications in patients with blunt multiple trauma with and without associated head trauma. DESIGN: Retrospective. SETTING: Level I trauma center. PATIENTS: Patients were included if they were treated between August 1987 and September 1995. Inclusion criteria included age between 16 and 65 years, injury severity score (ISS) of more than twenty points, and clinical reexamination performed more than three years after the initial injury. METHODS: The records of each patient were abstracted to determine the ISS, the Glasgow coma score (GCS), and parameters describing the course of intensive care. For each patient, a reexamination was performed between January and September 1998. Patients with multiple trauma and associated head trauma (Group PTH, polytrauma, GCS less than nine points, and head computed tomography scan abnormalities) and patients with multiple trauma without associated head trauma (Group PT, polytrauma, GCS of at least nine points, and normal head computed tomography scans) were compared. A clinical reexamination was performed to evaluate functional outcome. RESULTS: Sixty-four patients belonged to Group PTH and 124 patients belonged to Group PT. There were no differences in the age (Group PTH, 28.9 +/- 1.6 years; Group PT, 29.2 +/- 2.1 years) or severity of injury (ISS Group PTH, 31.0 +/- 5.3 points; ISS Group PT, 33.0 +/- 6.1 points) among patients in the two groups. The overall incidence of periarticular heterotopic ossification was comparable in patients with multiple trauma with and without head injury (Group PTH, 30 of 64 patients [46.9 percent]; Group PT, 53 of 124 patients [42.7 percent]). The duration of ventilation was significantly higher in Group PT (Group PTH, 9.3 +/- 2.4 days; Group PT, 14.2 +/- 3.1 days; p = 0.02). In the subgroups in which heterotopic ossification developed (PT-HO and PTH-HO), patients in PT-HO had a significantly higher incidence of heterotopic ossification, as compared with patients in PTH-HO at initially uninjured joints (Group PTH-HO, 1 of 30 patients [3.3 percent]; Group PT-HO, 10 of 53 patients [18.9 percent]; p = 0.04). CONCLUSIONS: There was a high incidence of heterotopic ossification around those joints that were initially classified as uninjured in patients without head trauma. This finding suggests that pathogenic pathways independent of head trauma, such as long-term ventilation, play a main role. Causative factors for the development of heterotopic ossification at initially uninjured joints in long-term ventilated patients with multiple trauma with and without head trauma remain to be elucidated.

Adult↗
PubMed2001

Head injury mechanisms in helmet-protected motorcyclists: prospective multicenter study.

BACKGROUND: In a prospective study, three research groups at Hannover (H) and Munich (M) in Germany and Glasgow (G) in the United Kingdom collected data from motorcycle crashes between July 1996 and July 1998 to investigate head injury mechanisms in helmet-protected motorcyclists. METHODS: The head lesions of motorcyclists with Abbreviated Injury Score-Head (AISHead) 2+ injuries and/or helmet impact were classified into direct force effect (DFE) and indirect force effect (IFE) lesions. The effecting forces and the force consequences were analyzed in detail. RESULTS: Two-hundred twenty-six motorcyclists (H, n = 115; M, n = 56; and G, n = 55) were included. Collision opponents were cars (57.8%), trucks (8.0%), pedestrians (2.3%), bicycles (1.4%), two-wheel motor vehicles (0.8%), and others (4.2%). In 25.4% no other moving object was involved. The mean impact speed was 55 km/h (range, 0-120 km/h) and correlated with AISHead. Seventy-six (33%) motorcyclists had no head injury, 21% (n = 48) AISHead 1, and 46% (n = 103) AISHead 2+. Four hundred nine head lesions were further classified: 36.9% DFE and 63.1% IFE. Lesions included 20.5% bone, 51.3% brain, and 28.1% skin. The most frequent brain lesions were subdural hematomas (22.4%, n = 47) and subarachnoid hematomas (25.2%, n = 53). Lesions of skin or bone were mainly DFE lesions, whereas brain lesions were mostly IFE lesions. CONCLUSION: A modification of the design of the helmet shell may have a preventative effect on DFE lesions, which are caused by a high amount of direct force transfer. Acceleration or deceleration forces induce IFE lesions, particularly rotation, which is an important and underestimated factor. The reduction of the effecting forces and the kinetic consequences should be a goal for future motorcycle helmet generations.

Accidents, Traffic↗
PubMed2001

Molecular cloning of a functional promoter of the human plakoglobin gene.

OBJECTIVE: Plakoglobin (Pg) is the only cytoplasmic protein component common to both junctional complexes mediating cell-cell adhesion, adherens junctions and desmosomes. In these complexes Pg appears to act as a linker protein anchoring transmembrane proteins of the cadherin superfamily to the actin cytoskeleton and intermediate filament system respectively. Intercellular adhesion is frequently disturbed in skin diseases and in carcinomas, enabling tumour progression and metastasis. Whereas Pg expression is lost in some thyroid tumours and carcinoma cell lines, little information on Pg gene regulation is currently available owing to a lack of promoter studies. DESIGN AND METHODS: We have cloned and sequenced genomic DNA from a human library that resulted in 979 bp upstream of the published Pg cDNA. The transcriptional start was mapped by rapid amplification of cDNA ends. Methylation-specific PCR of bisulfite-modified cell line DNA was applied to probe the methylation status of a promoter-associated CpG island. Reporter-gene constructs of various promoter fragments were transiently transfected in thyroid carcinoma cell lines and their activities were determined by luciferase measurements. RESULTS AND CONCLUSIONS: A 1 kb DNA fragment harbouring a functional promoter of the human Pg gene was cloned and characterized. The sequence lacks a canonical TATA box, but contains putative CCAAT boxes as well as various putative binding sites for transcription factors, among them SP1 and AP2, proximal to the transcriptional start. Considerable promoter activity was found in thyroid cell lines and deletion analysis indicated that a 300 bp region proximal to the 5'-untranslated region of the mRNA represents the minimal promoter of the human Pg gene. As cells lacking endogenous Pg expression were found to contain methylated CpG dinucleotides in a CpG island located around the transcriptional start site, it is suggested that epigenetic mechanisms such as DNA methylation contribute to dysregulated Pg expression.

5' Flanking Region↗
PubMed2001

[Promoter hypermethylation during tumor progression: quantitative analysis employing kinetic PCR].

The methylation of cytosine residues in the promoter region of tumour-suppressor genes is now widely recognised as an alternative mechanism of gene inactivation in cancer. To address the question whether promoter methylation is an early event in the process of malignant transformation and whether quantitative changes are occurring during clonal evolution we developed a new assay for the measurement of cytosine methylation, which could also be applied to laser-microdissected archival specimens. We first improved the bisulfite conversion of genomic DNA in order to analyse cells isolated from archival tissue sections by laser-assisted microdissection. Analysing the methylation of key regulatory genes in archival specimens (n = 38) we could demonstrate that aberrant promoter hypermethylation is an early event during breast cancer development and changes quantitatively and in a gene-specific manner during progression. This novel methodological approach allows the morphology-guided quantitative analysis of changes in the methylation pattern during the clonal evolution of tumour cells. Quantitative differences detected by this assay will offer new insights into early steps in carcinogenesis and tumour cell heterogeneity.

DNA Methylation↗
PubMed2000

Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction.

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.

Antigens, Neoplasm↗
PubMed2000

Common and epithelioid variants of hepatic angiomyolipoma exhibit clonal growth and share a distinctive immunophenotype.

Angiomyolipoma represents a rare liver tumor of uncertain histogenesis that is commonly considered a hamartoma. A series of 12 hepatic angiomyolipomas, including 3 samples of the epithelioid subtype, was analyzed for clonality using the human androgen receptor gene locus (HUMARA). Four of 6 informative cases revealed monoclonality. The polyclonal pattern in the 2 remaining cases was most probably caused by excessive infiltration of inflammatory cells. Monoclonality with an identical X-chromosomal inactivation pattern in all nodules was found in a multifocal recurrent tumor indicating a metastatic process. Despite the morphologic heterogeneity, all tumors displayed an identical immunohistochemical labeling pattern. It is concluded that different subtypes of hepatic angiomyolipoma exhibit a monoclonal and hence probably neoplastic growth and share an identical immunophenotype suitable for their identification even in small biopsy specimens. The epithelioid subtype may give rise to intrahepatic metastasis.

Adult↗
PubMed2000

Positive display of methylated sites: a novel method for the detection of promoter methylation.

Promoter methylation represents an important mechanism for silencing gene expression in higher eukaryotes. To study methylation of the promoter of the tumor suppressor p16INK4a, a fast and simple method was developed that, in contrast to previous studies, relies on the positive display of methylated sites (PDM). The method is based on bisulfite treatment of DNA, polymerase chain reaction (PCR)-amplification of the modified DNA and restriction digest of de novo created restriction sites to positively display DNA methylation in a background of unmethylated DNA. Since methylated as well as unmethylated DNA is amplified, information on the proportion of both is provided. Using this approach, 33 ductal invasive carcinomas, 4 normal mammary tissues, and 4 cell lines were analyzed for methylation. Methylation in the p16INK4a promoter was detected in 1 of 33 carcinomas (3%) and in 0 of 4 normal tissues. The conclusion is that PDM provides a useful tool in determining the degree and pattern of promoter methylation and is suitable to screen large series of tissue samples.

Base Pairing↗
PubMed2000

Charcot-Marie-Tooth neuropathy type 2 and P0 point mutations: two novel amino acid substitutions (Asp61Gly; Tyr119Cys) and a possible "hotspot" on Thr124Met.

Mutations in the gene for the major protein component of peripheral nerve myelin, myelin protein zero (MPZ, P0), cause hereditary disorders of Schwann cell myelin such as Charcot-Marie-Tooth neuropathy type 1B (CMT1B), Dejerine-Sottas syndrome (DSS), and congenital hypomyelinating neuropathy (CHN). More recently, P0 mutations were identified in the axonal type of CMT neuropathy, CMT2, which is different from the demyelinating variants with respect to electroneurography and nerve pathology. We screened 49 patients with a clinical and histopathological diagnosis of CMT2 for mutations in the P0 gene. Three heterozygous single nucleotide changes were detected: two novel missense mutations, Asp61Gly and Tyr119Cys, and the known Thr124Met substitution, that has already been reported in several CMT patients from different European countries. Haplotype analysis for the P0 locus proved that our patients with the 124Met allele were not related to a cohort of patients with the same mutation, all of Belgian descent and all found to share a common ancestor. Our data suggest that P0 mutations account for a detectable proportion of CMT2 cases with virtually every patient harbouring a different mutation but recurrence of the Thr124Met amino acid substitution. The high frequency of this peculiar genotype in the European CMT population is presumably not only due to a founder effect but Thr124Met might constitute a mutation hotspot in the P0 gene as well.

Adult↗
PubMed2000

Detection of gene amplification in intraductal and infiltrating breast cancer by laser-assisted microdissection and quantitative real-time PCR.

Gene amplification is one essential mechanism leading to oncogene activation which is supposed to play a major role in the pathogenesis of invasive breast cancer. However, using standard methodologies the detection of gene amplifications has been limited especially in small-sized lesions, like pre-invasive precursor lesions. The combination of two novel technologies, laser-based microdissection and quantitative real-time PCR, facilitates the detection of low-level amplifications in morphologically defined lesions. As a model system we investigated in situ breast cancer (ductal carcinoma in situ, DCIS) classified according to the morphology-based Van Nuys grading system for amplification of growth-regulatory genes. In this study 83 formalin-fixed, paraffin-embedded archival DCIS specimens were examined after laser-based microdissection by quantitative real-time PCR using the TaqMan detection system for amplification of the c-erbB2, topoisomerase IIalpha, c-myc and cyclinD1 gene. In a subset of 17 DCIS with adjacent infiltrating tumour components we compared intraductal and invasive tumour components in parallel for differences in amplification status. The combination of these new techniques represents an excellent tool to gain new insights into carcinogenesis by analyzing genetic alterations in morphologically identified heterogeneous lesions in breast cancer progression within the very same specimen or even tissue slide.

Antigens, Neoplasm↗
PubMed2000

Laser-assisted microdissection and short tandem repeat PCR for the investigation of graft chimerism after solid organ transplantation.

The detection of donor-derived cells in the blood and tissues of graft recipients after solid organ transplantation is a readily observed phenomenon called microchimerism. Yet very little is known about the persistence and integration of recipient-derived cells in the transplanted organ, indicating a form of intragraft chimerism. To further study this phenomenon and its possible influence on graft acceptance or rejection, we developed the following novel approach. Immunohistochemically labeled cells were isolated by means of laser-based microdissection and subsequent laser pressure catapulting from paraffine-embedded posttransplantation biopsies. The following use of a highly sensitive PCR assay analyzing one polymorphic short tandem repeat (STR) marker enabled us to clearly identify the genotypes in samples containing as little as 10 isolated cells. The combination of laser-based microdissection and STR-PCR thus provides a powerful tool for the genotyping of even very few cells isolated from routinely processed biopsies after solid organ transplantation.

Cell Separation↗
PubMed2000

Quantitative molecular analysis of laser-microdissected paraffin-embedded human tissues.

Laser microdissection enables the contamination-free isolation of morphologically defined pure cell populations from archival formalin-fixed paraffin-embedded tissue specimens. Cells isolated by this method have been characterized by a wide variety of qualitative molecular assays, e.g. loss of heterozygosity, point mutations, clonality and lineage origin. The recently introduced real-time PCR technology renders the reliable quantification of very small amounts of nucleic acids possible. Several groups including our own showed that this technique can be successfully applied for the quantification of DNA and RNA isolated from microdissected archival tissue sections, even after immunohistochemical staining. The exact analysis of quantitative changes of nucleic acids during the course of pathological alterations has thus become possible. In many situations these quantitative changes can be expected to be more important than qualitative changes. The new technology for the quantification of structural genomic alterations and changes in the gene expression pattern in conjunction with microdissection have equipped morphologists with a powerful tool to study reactive and neoplastic changes of tissues.

Cell Separation↗
PubMed1999

Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes.

To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes.

Animals↗
PubMed1999

Long term results after multiple injuries including severe head injury.

OBJECTIVE: To describe the long term results in patients with multiple injuries including severe head injury. DESIGN: Retrospective and prospective clinical study. SETTING: Level I trauma centre, Germany. PATIENTS: Patients aged 16-60 years who had been injured more than 2 years before, whose Injury Severity Score was over 20 and whose cranial Abbreviated Injury Score (AIS) was over 3. MAIN OUTCOME MEASURES: Glasgow Outcome Scale (GOS), functional, neuropsychological, vocational and social outcomes. RESULTS: 58 patients, median age 24 (range 16-53, interquartile range (IQR) 21-32) years were investigated 5 (3-9; IQR 4-7) years after their injury. Median ISS was 34 (21-57; IQR 26-41) and GCS 6 (3-8; IQR 4-7). Duration of coma was 10 (2-51; IQR 7-22) days and neurological rehabilitation lasted 169 (10-830; IQR 80-300) days. Movements of the elbow and ankle was most impaired by injury. All psychometric tests showed deficits, particularly in speed of processing, concentration, recent memory, and learning performance. The social environment had been changed in half and vocational rehabilitation was dependent on age. 24 (42%) returned to their former profession, 18 (31%) were retrained to another profession, 16 (27%) were unemployed or retired on a pension. 31 (53%) made a good recovery with moderate disability, 19 (33%) had severe disability, and 8 (14%) remained in a persistent vegetative state assessed by the GOS. CONCLUSION: Early and concentrated rehabilitation facilitates functional, social, and neuropsychological reintegration.

Adolescent↗