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K Zimmermann

Publications and source records attributed to K Zimmermann.

At least 55 records · Page 3Linked to original sources
PubMed1998

In unison: regularization of protein secondary structure predictions that makes use of multiple sequence alignments.

We present a method whose purpose is to post-process the fuzzy results of secondary structure prediction methods that use multiple sequence alignments, in order to obtain 'realistic' secondary structures, i.e., secondary structure elements whose length is greater than or equal to some predefined minimum length. This regularization helps with interpretation of the secondary structure prediction.

Algorithms↗
PubMed1998

[Quality control and documentation of effectiveness of bacterial autovaccines with the use of flow cytometry].

Autovaccines are bacterial preparations derived from non pathogenic autologous bacteria of human origin. In the course of microbiological therapy these individual bacterial vaccines are mainly used in conditions of chronic inflammatory disorders of the respiratory or gastrointestinal tract as well as allergic diseases. Being autologous bacterial preparations from the patients own flora the control of batch variabilities of these individual products represents a special challenge for the manufacturer. A flow cytometric method suitable for batch control of autovaccines is described. The method is based upon the determination of the de novo expression of the CD69 antigen on different leucocyte subpopulations in whole blood cultures after preincubation of cells with different batches of autovaccines. Thus, manufacturers of autovaccines and other microbial preparations are able to reliably control batch variability and immunological activity of such products in accordance with drug regulations. The results of this study highlight the pharmaceutical quality of the individual therapeutic agent autovaccine.

Antigens, CD↗
PubMed1998

Expression of p21WAF1 predicts outcome of esophageal cancer patients treated by surgery alone or by combined therapy modalities.

The p21WAF1 protein is an important regulator of the cell cycle. Its expression and prognostic significance were investigated immunohistochemically in samples of normal esophageal squamous epithelium (n = 10), severe squamous cell dysplasia (n = 20), carcinoma in situ (n = 14), permanent esophageal squamous cell carcinoma cell lines (n = 3), and invasive squamous cell carcinomas treated either by potentially curative resection (n = 172) or by combined modality therapy (radiochemotherapy +/- surgery; n = 38). Whereas p21WAF1 expression in the normal epithelium was restricted to a few cells adjacent to the basal cell layer, p21WAF1 overexpression was frequently found in preneoplasias and invasive carcinomas. Expression of p21WAF1 in invasive carcinomas was not correlated with tumor differentiation, pT category, or pN category. Among carcinomas treated by potential curative resection, univariate (P = 0.0025) and multivariate (P = 0.0081) survival analysis showed significant correlation of strong p21WAF1 expression (> or =50% p21WAF1-positive tumor cells) with poor overall survival. Univariate survival analysis (P = 0.0006) revealed the same prognostic influence in the group of patients treated by combined modality therapy. We conclude that overexpression of p21WAF1 protein is a frequent event in preneoplasias and neoplasias of the esophagus. Immunohistochemical examination of p21WAF1 expression may provide important prognostic information for decision-making in the treatment of patients with esophageal cancer.

Adult↗
PubMed1997

Detection of tick-borne encephalitis virus by sample transfer, plaque assay and strand-specific reverse transcriptase polymerase chain reaction: what do we detect?

Experimental inoculation of mice provides a well characterized model for studying infection with tick-borne encephalitis virus (TBEV), a flavivirus pathogenic for humans. Conflicting data on the kinetics of viremia and the development of virus titers in the brain, however, were only recently shown to have resulted from the use of assay systems with different levels of sensitivity in the titration of TBEV, i.e. plaque assay or sample transfer into naive recipient mice. Theoretically, RT-PCR could extend further the detectability to antibody-neutralized virus and when undertaken strand-specifically discriminate active replication from the mere presence of TBEV. We have compared the conventional methods for detection of TBEV with a newly devised RT-PCR method. As expected, RT-PCR, in contrast to the infectivity assays, detected antibody-neutralized virus. Furthermore, the mere presence or active replication of the virus could be differentiated by strand-specific RT-PCR. Plaque assay and sample transfer, in contrast, both detected only infectious virus. However, whereas sample transfer provides higher sensitivity for detection of TBEV from solid organs, the plaque assay is less costly and considering animals welfare more convenient. Thus, the newly devised method may allow the resolution of unanswered questions, while both the traditional infectivity assays retain their benefits in certain situations.

Animals↗
PubMed1997

Detection of false-negative results in nested primer PCR of proviral HIV-1 DNA.

A control template for a competitive nested primer PCR of the HIV-1 gag region was constructed. This construct shares the primer recognition sequences with the wild-type template and yields a 97-bp fragment after amplification (wild-type: 115 bp). To provide an internal control for the individual PCR runs, six copies of this nested primer control plasmid were introduced into a reaction tube containing the specific sample (under the PCR conditions used, this copy number reproducibly gave a positive PCR signal). The results of our study show the feasibility of this concept by analyzing a plasmid (pBH10) containing HIV-1 wild-type sequences, and examination of samples from a cohort of HIV-1-seropositive subjects demonstrated the clinical usefulness of this test. The control plasmid was detectable in all of the samples but one, which without the use of the control template would have yielded a false-negative result.

Binding, Competitive↗
PubMed1997

Tumor uptake and metabolism of copper-67-labeled monoclonal antibody chCE7 in nude mice bearing neuroblastoma xenografts.

METHODS: ChCE7, an internalizing, neuroblastoma-specific monoclonal antibody (MAb), and its F(ab')2 fragments were derived with the bifunctional ligand 4-(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl benzoic acid tetrahydrochloride (CPTA) and labeled with the potential therapeutic nuclide 67Cu. After internalization and degradation of these immunoconjugates in SKN-AS human neuroblastoma cells, the terminal degradation product was found to be the lysine adduct of the copper complex. In vivo distributions in nude mice bearing neuroblastoma xenografts were studied and extracts from tumor and tissue samples were analyzed. RESULTS: The intact MAb showed high tumor uptake, stable over 4 days postinjection (33.7% +/- 2.8% ID/g), with tumor/blood ratios increasing from 4.4 on Day 1 to 23.0 on Day 7 postinjection and low levels of radioactivity in other tissues. Analysis of tumor extracts by gel filtration chromatography and high-pressure liquid chromatography (HPLC) showed that over the period of 4 days radioactivity was present both in a high M(r) form, consisting of the MAb/antigen complex, as well as in a low M(r) form, consisting of the copper complex attached to short peptides, including the lys-CPTA complex. There was no evidence of aggregates or MAb/antigen complexes in the blood, radioactivity being exclusively in the form of intact MAb, and radioactivity in the liver was found to consist of intact MAb, MAb fragments and the lys-CPTA metabolite. In the case of the F(ab')2 fragments, high accumulation of radioactivity in the kidneys was observed and analysis of kidney extracts showed it to be due to rapid accumulation of the lys-CPTA complex. When kidney uptake and retention of the CPTA complex as well as of its lysine and glycine adducts was investigated, the lysine complex was taken up more strongly and retained longer in the kidneys than the other compounds. CONCLUSION: Copper-67-labeled MAb chCE7 F(ab')2 fragments were prepared using a novel bifunctional copper ligand 1-(p-aminobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (DO3A). Compared with MAb-chCE7 F(ab')2 fragments labeled by the CPTA ligand, labels using the DO3A ligand showed improved biodistributions resulting, 48 hr postinjection, in a 4-fold increase in tumor uptake and a 4-fold reduction of radioactivity in the kidneys.

Animals↗
PubMed1996

Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation.

The two-signal model of T-cell activation postulates that T lymphocytes require at least two distinct signals for activation. This model has been established with bulk cultures of T cells in which T-cell-T-cell interaction can occur, possibly delivering further unrecognized costimulatory signals. The signal requirements of single T cells for the induction of clonal cell growth or the transcription of cytokines would best be studied in a cell cloning system in the absence of feeder cells; however, such an experimental system has not been reported so far. In this study, we report the long-term cloning of human resting peripheral blood CD4+CD45RO- T cells under feeder cell-free conditions in response to CD3 and CD28 stimulation in the presence of exogenous interleukin-2 (IL-2). Cloning efficiency ranged from 40% to 60% depending on the presence of additional cytokines IL-1 and IL-6. Single-call polymerase chain reaction showed that transcription of IL-2 occurred in cells stimulated through CD3 plus CD28 alone. T cells grown in response to CD3 plus CD28 plus IL-2 stimulation produced both IL-4 and interferon-gamma (IFN-gamma) on restimulation (Th0 cells) and could be functionally differentiated into Th1- or Th2-type cells by the addition of IFN-gamma or IL-4, respectively, during cell cloning. These data show on the single-cell level a two-signal model of T- cell activation for the transcription of IL-2. In addition, these experiments show that IFN-gamma and IL-4 exert their T-cell-differentiating effects directly on the T cell without any further need for antigen-presenting cells. Together, our experiments show the feasability of a defined long-term clonal cell culture system to study the growth and differentiation of human T lymphocytes.

Antibodies, Monoclonal↗
PubMed1996

Allostimulated lymphocytes inhibit replication of HIV type 1.

Previous reports demonstrated that alloantigen- or xenoantigen-specific antibodies displayed neutralizing activity toward human or simian immunodeficiency viruses. In the present article we have addressed the question of alloantigen-induced cell-mediated anti-HIV activity. We show that allostimulation resulted in a lymphocyte population (largely of the CD8-positive phenotype) with the capacity to inhibit HIV-1 replication in PHA blasts of homologous and, unexpectedly, also autologous origin. The allostimulated effector cells exerted their activity via a noncytolytic mechanism. Experiments in which direct cell-to-cell contact between allostimulated effectors and HIV-1-infected PHA blasts was prevented by a semipermeable membrane indicated that soluble mediators were involved in inhibition of HIV-1 replication. As such allostimulated effectors not only would have the capacity to prevent viral replication in allogeneic HIV-1-infected cells (known to play an important role in HIV-1 transmission in vivo), but also might inhibit HIV-1 growth in autologous lymphocytes, the concept of an AIDS vaccine containing both HIV-1 antigens and alloantigens warrants further consideration.

Cell Line, Transformed↗
PubMed1996

[Measurement of expired alcohol concentrations with a new electrochemical sensor. A model investigation to determine interference with volatile anesthetics and clinical application].

UNLABELLED: Absorption of irrigating fluid in transurethral prostatic resection (TURP) and percutaneous nephrolitholapaxy (PNL) into veins or delayed absorption due to fluid extravasation may result in a TURP syndrome. The measurement of end-tidal breath alcohol concentration (et AC) as a monitor of absorption of irrigating fluid labelled with 2% ethanol is limited by the disturbance of infrared sensors by volatile anaesthetics and nitrous oxide (N2O) (Fig. 2). An electrochemical sensor is acceptable for this method. The aim of the present study was the evaluation of breath alcohol measurements using an electrochemical sensor device (Alcomed 3010, Envitec). The stability of the sensor in the presence of volatile anaesthetics was examined using a lung model. In a clinical investigation, the device was then applied to spontaneously breathing or mechanically ventilated patients inhaling volatile anaesthetics during endoscopic urological surgery. METHOD: A two-chamber lung model filled with water for performing noninvasive measurements at the mouth of a patient has already been introduced by Brunner et al. (Fig. 1). With the addition of different amounts of ethanol to the temperature-controlled water, a constant ethanol concentration is achievable in the air above the water that is dependent on adjustments of the ventilator. Increasing concentrations of volatile anaesthetics (isoflurane, enflurane, halothane, and sevoflurane) were added to the fresh gas flow (2 l O2/3 l N2O) and etACs were measured using the manually triggered self-absorbent electrochemical sensor. First, regression equations were established between breath alcohol concentrations and increased volatile anaesthetic concentrations. Regression equations were then established between end-tidal anaesthetic gas concentrations and vaporizer adjustments in order to rule out an influence of ethanol on the anaesthetic gas monitor Ultima V (Datex). In the clinical investigation, 53 intubated and ventilated patients (33 undergoing PNL, 20 undergoing TURP) and 48 patients breathing spontaneously (32 with inhalation anaesthesia, 16 with spinal anaesthesia) were investigated. The etAC was measured with the Alcomed 3010 and compared with gas-chromatographically registered blood alcohol concentrations (BAC). The study had previously been approved by the Ethical Committee of the Medical University of Luebeck. Patients with liver disease and a history of toxic abuse were excluded. Only one value per patient (maximum BAC) was included in the statistics in order to avoid a cluster effect. RESULTS: The lung model experiments demonstrated that the measurement of etAC with an electrochemical sensor is free of interference by volatile anaesthetics (Table 1). The slope of the regression between the measured alcohol concentration and increased concentrations of anaesthetics did not differ significantly from baseline values. The measurement of end-tidal anaesthetic concentrations was not significantly different from vaporizer adjustments in the presence of increased alcohol concentrations (Table 2). During the clinical investigation, a regression between etAC and BAC was determined for both groups. For the group of patients breathing spontaneously, the correlation coefficient was 0.961 and the regression equation revealed etAC = 0.5677*BAC-0.1303 (Fig. 5). However, in the group of ventilated patients a biphasic course was shown that was dependent on BAC (Fig. 6). At BAC < 0.4%, a similar correlation (r = 0.856) to the spontaneously breathing group could be seen (regression equation: etAC = 0.617*BAC-0.020). Above 0.4% BAC there was no acceptable correlation (r = 0.444, regression equation: etAC = 0.202*BAC+0.104). CONCLUSIONS: The tested electrochemical sensor does not interfere with volatile anaesthetics and N2O as demonstrated by a lung model. There is a good correlation between etAC and BAC measurements in patients breathing spontaneously with special regard to the slope of the regression (s = 0.57).

Anesthetics↗
PubMed1996

Effect of high- versus intermediate-purity blood coagulation factor concentrates on HIV-1 replication.

Controversy exists as to whether treatment of HIV-1-seropositive hemophiliacs with blood coagulation products of high purity might help prevent the decline of CD4-positive lymphocytes and thus delay progression toward AIDS. As viral load has recently been shown to be associated with disease progression in HIV-1 infection, we tested for a possible direct interference of high- or intermediate-purity blood coagulation products with replication of HIV-1. The data obtained revealed comparable replication of HIV-1 in the presence and absence of all blood coagulation products tested (assessed by PCR-based quantitation of proviral HIV-1 DNA in infected cells after a 10-day incubation period). These data suggest that coagulation factor concentrates per se will also have no direct effect on HIV-1 replication in vivo.

CD4 Lymphocyte Count↗
PubMed1996

Quantitative multiple competitive PCR of HIV-1 DNA in a single reaction tube.

A quantitative multiple competitive PCR (QMC-PCR) for determination of DNA copy numbers is described. Four competitive DNA templates for the env region of HIV-1 were constructed with sizes longer (187 and 163 bp) or shorter (122 and 105 bp) than the 142 bp of the wild-type PCR product. Varying amounts of each of these competitors are introduced together with the sample into a single reaction tube. Since competitors and wild-type fragments share the same primer recognition sequence (SK68/SK69), amplification occurs according to the rate of the introduced copy numbers. The PCR products are run on an agarose gel, and the copy number of the sample is determined by analyzing the bands with a video densitometer and calculating the equivalence point in a linear regression plot.

Binding, Competitive↗
PubMed1995

Genomic sequences and structural organization of the human nidogen gene (NID).

Nidogen/entactin is a ubiquitous 150-kDa multidomain basement membrane protein. Since in vitro binding studies indicated that nidogen may function as a major mediator in basement membrane organization and assembly, analysis of gene structure and regulation of gene expression will help us to understand many biological processes that involve degradation and reorganization of the basement membrane zone. An approximately 100-kb region of genomic DNA encoding the human nidogen gene (NID) including 5' and 3' flanking sequences has been cloned and characterized by restriction mapping and sequencing. The entire gene is more than 90 kb in length and contains 20 exons. All introns interrupt protein coding sequences. The size of individual introns varies significantly, ranging from 0.6 to 18 kb. Its exon/intron structure revealed that the protein domains of human nidogen are organized in a domain-specific manner with various subdomains being encoded by individual exons, indicating that exon duplication and shuffling have played an important role in determining the present structure of the protein. Comparison of the exon organization with the recently published ascidian nidogen amino acid sequence strongly suggests that vertebrate nidogen might have evolved from a common ancestral precursor resembling ascidian nidogen.

Base Sequence↗
PubMed1995

Binding properties and protease stability of recombinant human nidogen.

Recombinant human nidogen was obtained from transfected kidney cell clones as a 150-kDa protein with a three-globule structure. It was modified by sulfation and O-glycosylation and a lower level of N-glycosylation than mouse nidogen. Recombinant nidogens of both species were, however, indistinguishable in their affinities for laminin-1 and a recombinant laminin gamma 1 chain fragment and showed a similar binding to collagen IV and the heparan sulfate proteoglycan perlecan. The two nidogens were also equivalent in the promotion of ternary complex formation between these ligands, indicating that this function has been conserved during mammalian evolution. Fewer zinc-binding sites could be identified in human nidogen and correlated with a lower capacity of zinc to prevent binding to laminin and collagen IV. Most remarkable was the greater sensitivity of human nidogen to endogenous proteolysis in cell culture, yielding fragments of 90-145 kDa. Studies with several exogenous proteases, including thrombin and leucocyte elastase, showed lack of stability of the N-terminal globular domain G1 in contrast to what was found for mouse nidogen. Since such degradation could be important for basement membrane remodelling, this difference between human and mouse may be biologically significant.

Amino Acid Sequence↗
PubMed1995

Cellular processing of copper-67-labeled monoclonal antibody chCE7 by human neuroblastoma cells.

Monoclonal antibody chCE7, an internalizing neuroblastoma-specific chimeric antibody, was derivatized with the macrocyclic amine ligand 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid tetrahydrochloride and labeled with the potential therapeutic nuclide 67Cu. Using pulse labeling and an acid elution endocytosis assay, 67Cu-chCE7 was found to be internalized into human neuroblastoma (SKN-AS) cells at a similar rate and to a similar extent as 125I-labeled chCE7. Uptake of 67Cu-chCE7 and 125I-chCE7 into the acid stable (intracellular) pool proceeded with similar kinetics during the first 2 h of internalization. However, in contrast to 125I-chCE7-loaded cells, at later times intracellular radioactivity kept increasing in the case of 67Cu-chCE7-loaded cells. It was shown that this effect is due to the intracellular accumulation of a low M(r) degradation product consisting of the 67Cu-4[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl] benzoic acid complex, possibly with a short peptide attached to it. Degradation of both 125I-chCE7 and 67Cu-chCE7 was inhibited by chloroquine, indicating endosomal or lysosomal degradation, and a 43,000 M(r) fragment was found to be the major high M(r) degradation product in both cases. Although at times between 4 and 6 h of internalization intracellular breakdown of 67Cu-chCE7 was found to proceed more slowly, the major difference between the two immunoconjugates resides in the prolonged cellular retention of the 67Cu-chCE7 metabolite.

Antibodies, Monoclonal↗