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Biomedical subjects

M Neumaier

Publications and source records attributed to M Neumaier.

At least 19 recordsLinked to original sources
PubMed2001

Human endostatin inhibits growth of human non-small-cell lung cancer in a murine xenotransplant model.

Overall prognosis in human NSCLC remains poor. Antiangiogenic treatment has become a promising concept for the treatment of solid malignancies. Our purpose was to evaluate the efficacy of recombinant HSENDO for the treatment of human NSCLC in an orthotopic murine xenotransplantation model. The efficacy of HSENDO was tested in vitro in cell-proliferation, cell-migration and tube-formation assays. In vivo, the effect of HSENDO on tumor growth was tested in s.c. xenotransplanted human NSCLC and on intrapulmonary induced human NSCLC. In vitro, HSENDO inhibited both human and rodent endothelial cell proliferation in a time- and dose-dependent fashion. Endothelial cell migration was inhibited by 97%. Tube formation of murine endothelial cells was inhibited and preexisting tubes degenerated after HSENDO exposure. In vivo, HSENDO delayed growth of s.c. xenotransplanted tumors. Immunohistochemic staining demonstrated no change in microvessel density but a significant reduction of proliferating tumor cells and an increase in bFGF and VEGF expression, reflecting the antiangiogenic effect of HSENDO. Intrapulmonary tumor induction caused death subsequent to metastatic disease. Systemic HSENDO application extended survival significantly. HSENDO was demonstrated to inhibit endothelial cell proliferation, migration and tube formation effectively. In vivo growth of s.c. transplanted tumors was delayed and survival extended by 32% and 69%, respectively, after intrapulmonary NSCLC induction.

Animals↗
PubMed2001

Fusion proteins of B7.1 and a carcinoembryonic antigen (CEA)-specific antibody fragment opsonize CEA-expressing tumor cells and coactivate T-cell immunity.

Genetic engineering can be used to generate antigen-specific molecules for improved tumor immunotherapy. We have constructed genes coding for fusion proteins consisting of a high-affinity antibody single-chain antibody fragment (scFv) specific for the human carcinoembryonic antigen (CEA) and the costimulation domain of the murine B7.1 molecule (mB7.1) linked to the antibody moiety by an IgG3 peptide linker. The hybrid genes were constructed in 2 orientations, one with the scFv located N-terminal to mB7.1 and one vice versa. Soluble proteins were expressed by CHO cells, purified using anti-idiotype-affinity chromatography and characterized by tumor-cell binding and costimulation activity. When tumor cells expressing CEA on the cell membrane were opsonized with the CEA-specific costimulators, both fusion proteins specifically stimulated murine T-cell preparations to proliferate in a similar manner. Our data suggest that "costimulation coating" of tumor cells may be a suitable approach for activation of a sustained cellular antitumor response. It also provides the opportunity to increase tumor immunogenicity using easily generated soluble fusion proteins that advantageously link biological functions of both the humoral and the cellular arm of the specific immune system.

Animals↗
PubMed2001

Detection of micrometastasis through tissue-specific gene expression: its promise and problems.

The detection of micrometastasis holds great promise for earlier staging of patients with malignant diseases and may ultimately guide therapeutic decisions. So far, reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of genes expressed by the tumor in a tissue-specific manner is the method with the highest diagnostic sensitivity. It is well-established that the identification of single tumor cells is feasible in tissues and bodily fluids in both experimental and clinical samples. However, at present it is difficult to assign clinical significance to results obtained from such tests, primarily because their diagnostic specificity is disputed, both conceptionally and methodologically. For example, amplification of candidate mRNA targets is detectable in non-cancer patients using conditions that generally fail to generate such signals from healthy individuals. We have established that transcription of the tissue-specific genes can be affected by different means. Specifically, some target mRNA species are detectable in peripheral blood nuclear cells as low abundance constitutive-like expression, whereas others are induced through in vitro tissue culturing. In addition, mRNA expression may be distinctly upregulated by different cytokines or growth factors in vivo. Also, background transcription of target mRNAs can occur in different lineages of peripheral blood cells. Finally, expression may be substantially different in tissues such as peripheral blood, bone marrow, or lymph nodes. As a consequence, cancer patients in unrelated clinical situations may present with different levels of background expression, making the diagnostic specificity of test results difficult to assess. To add to this complexity, an increasing body of literature is being generated using various targets for a multitude of malignant diseases. There is a great variety of methods for sampling, specimen processing, nucleic acids recovery, test conditions, and readout formats, making it impossible to compare data. In summary, modalities of quantitative RT-PCR methods and standardization issues should be discussed to address these questions.

DNA, Neoplasm↗
PubMed2001

C-reactive protein in patients who had operative fracture treatment.

C-reactive protein as an indicator of infection, may help to detect surgical complications early and provide a better outcome for patients. To obtain a baseline for the use of C-reactive protein, the kinetics of C-reactive protein levels of 330 patients who had operative fracture treatment were studied before and after surgery. All patients who had an uneventful postoperative course had similar evolution in their C-reactive protein values: the peak level, which occurred on the second postoperative day, depended on the region of trauma (femoral fractures, 15.4 mg/dL versus ankle fractures, 3.5 mg/dL) and reflected the extent of surgical trauma. Of 47 patients with complicated courses, C-reactive protein proved helpful as a marker in risk stratification and as an early indicator for infection. Of nine patients with a deep wound infection, a high rise of C-reactive protein was recorded, and seven patients showed a rise in the C-reactive protein level before the onset of clinical symptoms. A cut-off level of 14 mg/dL on the fourth day after surgery was recorded for the patients with deep wound infection.

Adolescent↗
PubMed2001

Controlled ribozyme targeting demonstrates an antiapoptotic effect of carcinoembryonic antigen in HT29 colon cancer cells.

PURPOSE: Clinical studies suggest that carcinoembryonic antigen (CEA) is associated with metastatic progression of colon cancer. However, the biological function of CEA is not well understood. We have established an approach that allows studying of CEA function within the intact pathophysiological context of human colon cancer cells. EXPERIMENTAL DESIGN: We expressed CEA-targeted ribozymes under control of a tet-off promoter system in human HT29 colon cancer cells. This approach allows regulation of CEA levels on the mRNA and protein level by 50% and enables screening analysis of CEA-mediated changes of gene expression by cDNA microarray analysis. RESULTS: Comprehensive analysis of 273 genes revealed that CEA affects expression of various groups of cancer-related genes, in particular cell cycle and apoptotic genes. Although cell cycle gene expression showed a balanced bidirectional dysregulation, apoptotic genes were unidirectionally down-regulated by CEA. In parallel phenotypic studies, CEA did not affect cell cycle or proliferation rate. However, CEA significantly protected HT29 cells from undergoing apoptosis under various conditions, including confluent growth, UV light, IFN-gamma treatment, and treatment with 5-fluorouracil. CONCLUSIONS: Our study suggests that CEA has an important regulatory role in apoptosis, and we propose that CEA is a survival factor for colon cancer cells.

Antineoplastic Agents↗
PubMed2000

CCKB/gastrin receptors mediate changes in sodium and potassium absorption in the isolated perfused rat kidney.

BACKGROUND: To evaluate the function of cholecystokinin B (CCKB)/gastrin receptors in the rat kidney, we identified the receptors by Northern blot and localized the receptors by immunohistochemistry. The functional effects of gastrin were studied under standardized in vitro conditions using the isolated perfused kidney. METHODS: Rat kidneys were mounted in an organ bath by attaching the renal artery to a perfusion system. A catheter was inserted into the renal vein and the ureter to collect samples that were analyzed for the concentrations of electrolytes. After a preperfusion period, gastrin-17-I was given via the renal artery (10-8 to 10-6 mol/L). Subsequently, hemodynamic parameters (for example, perfusate flow) and changes in sodium and potassium absorption were determined. All data were subjected to a nonparametric analysis of variance and, in case of significant results, to subsequent paired comparisons by the a posteriori Wilcoxon test. RESULTS: Northern blot analysis detected CCKB receptor transcripts in total RNA isolated from kidneys. Immunohistochemistry localized CCKB receptors on tubules and collecting duct cells. Compared with controls, gastrin (10-6 mol/L) caused a decrease in the fractional sodium reabsorption (basal 80%, 10 minutes after application of gastrin 71%, after 20 minutes 62%, P < 0.05). This effect was inhibited by the CCKB receptor antagonist L-365,260. Gastrin decreased urinary potassium excretion at 10-8 and 10-6 mol/L [maximal decrease at 10-6 mol/L from baseline values (100%) to 49% after 10 minutes and to 69% after 20 minutes, P < 0.05, N = 6]. This effect was also abolished by the CCKB receptor antagonist L-365,260. Gastrin (10-6 mol/L) reduced perfusate flow by 31% (P < 0.05). CONCLUSIONS: CCKB receptors are expressed in the rat kidney on tubules and collecting ducts. These receptors mediate changes in renal potassium and sodium absorption. In addition, gastrin causes a decrease in perfusate flow, indicating that CCKB receptors might also modulate vascular resistance in the kidney.

Animals↗
PubMed2000

Experiences with external quality assessment (EQA) in molecular diagnostics in clinical laboratories in Germany. Working Group of the German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM).

The German Societies for Clinical Chemistry (DGKC) and Laboratory Medicine (DGLM) have established an official working group on molecular diagnostics in the field of laboratory medicine. The group's objectives are to support the establishment of molecular biology methods for the use in diagnostics in German clinical laboratories. Towards this end, we have defined specific aims, which are 1) the implementation and extension of external quality assessment (EQA) schemes and methodological exercises offered to clinical diagnostic laboratories, 2) the establishment of a proficiency network and data base within the societies, 3) the implementation of an educational program in molecular diagnostic procedures for clinical chemists and laboratory physicians through the organisation of symposia and workshops and 4) the cooperation with other DGKC/DGLM working groups on shared aspects of laboratory analysis, e.g. preanalytics. The focus of this presentation is to introduce some of these goals in more detail with particular emphasis on the first two program aspects and to discuss experiences with these activities.

Clinical Laboratory Techniques↗
PubMed2000

Quantitative PCR.

The classic molecular biology methods like Northern or Southern blot analyse non-amplified DNA or RNA, but need large amounts of nucleic acids, in many instances from tissues or cells that are heterogeneous. In contrast, polymerase chain reaction (PCR)-based techniques allow us to obtain genetic information through the specific amplification of nucleic acid sequences starting with a very low number of target copies. These reactions are characterized by a logarithmic amplification of the target sequences i.e. increase of PCR copies followed by a plateau phase showing a rapid decrease to zero of copy number increment per cycle. Accordingly, the amount of specific DNA product at the end of the PCR run bears no correlation to the number of target copies present in the original specimen. However, many applications in medicine or research require quantification of the number of specific targets in the specimen. This has generated a rapidly increasing need for the development of quantitative PCR techniques. Prominent examples are the determination of viral load in blood specimens for the diagnosis of HIV or HCV infections, the determination of changes in gene dosage through amplification or deletion e.g. of MDR-1, erb-B2, c-myc or the loss of heterozygosity in general. Finally, the analysis of gene expression on the mRNA level does require quantitative approaches to reverse transcriptase PCR, e.g. for studies in morphogenesis or the profiling of cancer cells. Recent advances in technology allow detection of the increment per cycle of a specifically generated PCR product in "real-time mode". Together with the new powerful methods to dissect heterogeneous tissues or fractionate bodily fluids, this now sets the stage for a detailed analysis not only of the genes and genetic changes within a single cell, but also of the use such cell makes of its genes e.g. in pharmacogenomics. Examples of recent developments of the technology and their applications will be given.

Gene Amplification↗
PubMed1999

Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes.

The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14). The use of Ficoll gradients to separate nucleated cells completely restored the specificity of this CK20 RT-PCR assay. The CK20-expressing cells are positively identified to belong to the granulocyte fraction of leucocytes, which appear to express the gene on a background level. Our results demonstrate for the first time that CK20 mRNA expression is not limited to epithelium. Its occurrence in normal granulocytes has to be considered in tests designed to detect circulating cancer cells or micrometastases.

Biomarkers, Tumor↗
PubMed1998

Kinetic and affinity constants of epitope specific anti-carcinoembryonic antigen (CEA) monoclonal antibodies for CEA and engineered CEA domain constructs.

BACKGROUND: Carcinoembryonic antigen (CEA) is a human tumor antigen with the domain structure N-A1-B1-A2-B2-A3-B3, in which each domain is predicted to have an Ig-like fold and is known to bind epitope specific anti-CEA antibodies. OBJECTIVE: To determine the affinity constants of several domain specific anti-CEA antibodies using purified recombinant or synthetic domains. RESULTS AND CONCLUSION: We have determined the kinetic and affinity constants of several anti-CEA antibodies for CEA, CEA domains (A3-B3) expressed in HeLa cells, and a synthetic peptide corresponding to the A3 domain using a BIAcore biosensor. There was no difference in affinity for CEA among a murine (mT84.66), a mouse/human chimeric form (cT84.66) or a disulfide deleted version (delta SScT84.66) of this antibody. There was less than a five-fold drop in affinity of murine T84.66 for the A3-B3 domain expressed in HeLa cells compared to CEA. The synthetic A3 domain had an affinity constant for mT84.66 which was ten-fold less than for CEA. The affinity constants for CEA with several other anti-CEA monoclonal antibodies, including three antibodies which have almost identical CDR sequences (CEA.281, CEA.11 and CEM231) were also determined. CEM231 which had a two-fold higher affinity constant for CEA than either CEA.281 or CEA.11 had a two-fold faster on-rate which accounts for its higher affinity constant. This difference may be due to one or more of the amino acid differences present in H1 (N vs. S or D) and H3 (A vs. V).

Amino Acid Sequence↗
PubMed1998

Specificity of reverse transcriptase polymerase chain reaction assays designed for the detection of circulating cancer cells is influenced by cytokines in vivo and in vitro.

Several reverse transcriptase polymerase chain reaction (RT-PCR) assays have been described for the detection of circulating tumour cells in blood and bone marrow. Target mRNA sequences for this purpose are the cytokeratins (CK) 19 and 20, the carcinoembryonic antigen (CEA), and the prostate-specific antigen messages. In this study, we investigated biological factors influencing the specificity of the CK19 and CEA RT-PCR assays. Bone marrow, granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cells and peripheral blood samples obtained from healthy volunteers (n = 15; CEA n = 7), from patients with epithelial (n = 29) and haematological (n = 23) cancer and from patients with chronic inflammatory diseases (n = 16) were examined. Neither CEA nor cytokeratin 19 messages could be amplified from bone marrow samples from healthy subjects and from patients with haematological malignancies. In contrast, specimens from patients with inflammatory diseases scored positive up to 60%. To investigate the influence of inflammation on target mRNA expression, haemopoietic cells were cultured with and without cytokine stimulation in vitro. CK19 messages could be easily detected in cultured marrow cells without further stimulation, CEA messages only after gamma-interferon (gamma-INF) stimulation. In contrast, G-CSF-mobilized peripheral blood stem cells were positive for CK19 messages only after stem cell factor (SCF) or interleukin stimulation. We conclude that transcription of so-called tissue-specific genes is inductible in haemopoietic tissues under certain conditions. These factors have to be considered in future applications of RT-PCR for the detection of minimal residual disease.

Abdominal Neoplasms↗
PubMed1998

External quality assessment of molecular biology-based methods used in laboratories of clinical chemistry and human genetics.

The Reference Institute of Bioanalysis of the German Society of Clinical Chemistry has performed the first external assessment of molecular genetics methods used in medical diagnosis. The following procedures were tested: (I) DNA preparation from whole blood, (II) PCR amplification using "standard" primers, and (III) submarine agarose gel electrophoresis. Out of 50 participants, 45 returned samples for evaluation.

Chemistry, Clinical↗
PubMed1998

Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques.

The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing--resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end. We address aspects that should be considered when structuring a new molecular diagnostic laboratory, and we discuss individual preanalytical and analytical procedures, from sampling to evaluation of assay results. In addition, different means of controlling contamination are discussed. Because the methodology is in constant change, no general standards can be defined. Accordingly, this publication is intended to serve as a recommendation for good laboratory practice and internal quality control and as a guide to troubleshooting, primarily in amplification techniques.

Chemistry, Clinical↗
PubMed1997

Expression of CD66a (human C-CAM) and other members of the carcinoembryonic antigen gene family of adhesion molecules in human colorectal adenomas.

Among the members of the carcinoembryonic antigen (CEA) family, CD66a (human C-CAM) and CGM2 (CEA gene family member 2) mRNAs are frequently down-regulated in colorectal cancer. In contrast, nonspecific cross-reactive antigen (NCA) mRNA is overexpressed in the majority of these carcinomas. In animal models, the rodent homologues of CD66a have been shown to act as tumor suppressors, suggesting an important role in carcinogenesis. Here we investigate the mRNAs of CD66a, CGM2, and NCA in 22 human colorectal adenomas and the respective normal mucosa specimens by Northern blots. The expression of both CD66a and CGM2 changed in a concomitant fashion. Using oligonucleotides specific for the N-terminal domains, two CD66a transcripts 3.9 and 1.5 kb in size were identified. These showed a greater than 50% down-regulation in 20 of 22 and 18 of 22 adenomas, respectively. Reduction of the CGM2 message was observed in 21 of 22 cases. Complete or near-complete losses of the CD66a 3.9-kb mRNA and the CGM2 message were found in 13 of 22 and 15 of 22 of the tumors, respectively. The medians of CD66a and CGM2 expressions were between 0.3 and 0.0, respectively. The tumor:normal ratio of NCA mRNA expression was increased up to 2.4-fold in 11 of 22 adenomas. Altogether, these results compare well to the changes reported previously for colorectal carcinomas. The high frequency and early appearance of dysregulation of members of the carcinoembryonic antigen family during colorectal tumorigenesis suggests that these changes may be important for the development of the malignant phenotype.

Adenoma↗
PubMed1997

Quality management and influential factors for the detection of single metastatic cancer cells by reverse transcriptase polymerase chain reaction.

The sensitive and specific detection of micrometastasis holds great promise for earlier staging of cancer patients. By amplification of tissue-specific gene expression, the reverse transcriptase polymerase chain reaction (rtPCR) readily detects single tumour cells in different tissues. An increasing number of rtPCR assays with possible relevance for routine laboratory diagnostic procedures is currently being reported in the literature. Interestingly, when used in the clinical setting, assays for the same target mRNA perform very differently, despite comparable sensitivities and specificities in-vitro. Using rtPCRs specific for carcinoembryonic antigen (CEA), prostate-specific antigen (PSA) and cytokeratin 18 (CK 18), we have started to systematically investigate, both experimentally and in clinical specimens, a number of factors that contribute to the varying and seemingly implausible test results. Here we have concentrated on sample collection modalities, assay stability and test reproducibility at the sensitivity limit. Our results demonstrate in detail that, at the maximum sensitivity required for micrometastasis detection, preanalytical and statistical influences increasingly become important for the consistency of the assay results. We conclude that the prerequisite for translating the results from highly sensitive and specific rtPCR assays into clinically relevant data is the thorough definition of assay procedures and the number of tests performed on a sample. Addressing questions of standardization and quality control management is a central aspect yet to be emphasized in assay development and application of routine laboratory rtPCR tests in oncology.

Carcinoembryonic Antigen↗