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Biomedical subjects

K Zimmermann

Publications and source records attributed to K Zimmermann.

At least 19 recordsLinked to original sources
PubMed1999

Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element.

Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms.

Adult↗
PubMed1999

Irrigating fluid absorption during percutaneous nephrolithotripsy.

BACKGROUND: The most common complication during percutaneous nephrolithotripsy (PNL) is the destruction of organ structures with extravasation of the irrigation fluid into the retroperitoneal space. Consequently, there is an increased risk of a urosepsis and a complicated therapeutic course. In this study we aimed to show that extravascular absorption could be differentiated from intravascular absorption due to their unique absorption characteristics, and that these characteristics enable a prediction of possible post-operative complications. METHODS: In a prospective study of 31 patients with PNL, ethanol was added to the irrigating fluid and blood ethanol concentration (BEC) was measured by gas chromatography during the endoscopic procedure and in the recovery room. Following the guidelines of Hahn, patients were divided into two groups: group EVA, in whom extravasation had occurred with subsequent absorption; group IVA, those with intravascular absorption. Patients' post-operative progress along with diagnoses of renal perforations or bleeding, or signs of infection or sepsis, were comprehensively listed. RESULTS: EVA was diagnosed in 19 cases, and IVA in 12 cases. Maximum BEC levels were achieved after 20 min (median) in the IVA group, and 75 min in the EVA group (P < 0.05). Apart from their significantly higher demand for opioids (P < 0.05), EVA patients had been hospitalised for a substantially and significantly longer period of time (P < 0.01). Although without statistical significance, there was a higher rate of peri-operatively confirmed complications and prolonged intensive therapeutic treatment in the extravasation group. CONCLUSION: Retroperitoneal extravasation can be identified by using ethanol monitoring during and after PNL. Afflicted patients require considerably longer hospitalisation, probably because of the additional injury to surrounding organ structures.

Absorption↗
PubMed1998

Glyceraldehyde-3-phosphate dehydrogenase, the putative target of the antiapoptotic compounds CGP 3466 and R-(-)-deprenyl.

R-(-)-Deprenyl (Selegiline) represents one of the drugs currently used for the treatment of Parkinson's disease. This compound was shown to protect neurons or glias from programmed cell death in a variety of models. The mechanism of action of neuroprotection as well as inhibition of apoptosis remains elusive. CGP 3466 is a structurally related analog of R-(-)-deprenyl that exhibits virtually no monoamine oxidase type B inhibiting activity but is neuroprotective in the picomolar concentration range. We showed specific binding of CGP 3466 to glyceraldehyde-3-phosphate dehydrogenase by affinity binding, by affinity labeling, and by means of BIAcore(R) technology. Apoptosis assays based on the human neuroblastoma cell line PAJU established the importance of this interaction for mediating drug-induced inhibition of programmed cell death.

Animals↗
PubMed1998

Comparison of early events during infection of human and chimpanzee peripheral blood mononuclear cells with HIV-1.

Differences in early events during entry and integration of HIV-1 into peripheral blood mononuclear cells (PBMC) might contribute to the absence of AIDS-like disease in chimpanzees as compared to humans. To address this question, we first tested the in vitro susceptibility of human and chimpanzee PBMC for infection with the two HIV-1 isolates III B and RF. The results of these studies revealed that chimpanzee PBMC had a slightly lower capability to support the growth of HIV-1 as compared to human PBMC. This was accompanied by a delayed accumulation of proviral HIV-1 DNA in cultures of HIV-1-infected chimpanzee PBMC. However, no differences between cells of the two species were observed when very early events of HIV-1 infection were studied. Shortly (20 h) after infection chimpanzee and human cells harbored similar amounts of proviral HIV-1 DNA and PBMC of both species behaved comparably with respect to pre-integration latency (i.e. the ability to activate extrachromosomal HIV-1 intermediates in HIV-1 infected quiescent cells at various times after infection). These results strongly suggest that the absence of AIDS-like disease in chimpanzees cannot be correlated with defects in early events of the HIV-1 replicative cycle.

Animals↗
PubMed1998

Influence of ciprofloxacin and other antimicrobial drugs on different Escherichia coli strains in continuous-flow cultures under aerobic and anaerobic conditions.

In continuous-flow culture, long generation times and high bacterial counts favour survival of bacteria. A chemotherapeutic agent that achieves a bactericidal effect under these circumstances can therefore be seen as highly effective. In our continuous-flow culture we obtained bactericidal effects with ciprofloxacin 1-2 mg/L, cefotaxime 4 mg/L and mezlocillin 32 mg/L. These effects were seen irrespective of whether conditions were aerobic or anaerobic. There were no significant differences between monocultures and mixed cultures simulating faecal flora with the various Escherichia coli strains tested. Cefotaxime had an initial effect but an increase in counts was then observed as a result of regrowth of E. coli survivor strains in aerobic monoculture and mixed cultures. Mezlocillin was completely bactericidal in monocultures, but regrowth occurred in mixed cultures under anaerobic conditions. Neither the bacterial composition of this culture nor the resistance pattern explained this regrowth. These results were observed in long-term experiments followed for up to 7 days. We conclude that the antibiotics tested are highly effective against E. coli under unfavourable conditions simulating in-vivo situations.

Aerobiosis↗
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↗