Showing papers by "Humboldt University of Berlin published in 1997"

Monocyte deactivation in septic patients: restoration by IFN-gamma treatment.

Abstract: Neutralization of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1), decreases mortality in several animal models of sepsis. However, recent clinical trials did not show an unequivocal improvement in survival. In contrast to animals, which succumb to shock during the first 72 hours, we found that many patients die much later with signs of opportunistic infections accompanied by downregulation of their monocytic HLA-DR expression and reduced ability to produce lipopolysaccharide (LPS)-induced TNF-α in vitro1–3. This phenomenon of monocyte deactivation in septic patients with fatal outcome shows similarities to experimental monocytic refractoriness induced by LPS desensitization or by pretreatment with its endogenous mediators IL-10 and transforming growth factor-β (TGF-β)4. In order to strengthen their antimicrobial defense, here we tested whether interferon-γ (IFN-γ) can improve monocytic functions in these patients and in experimental monocytic deactivation. The considerably lowered in vitro levels of LPS-induced TNF-α in these situations were significantly enhanced by IFN-γ, but did not reach the extremely high levels of IFN-γ primed naive cells from healthy donors. Moreover, IFN-γ applied to septic patients with low monocytic HLA-DR expression restored the deficient HLA-DR expression and in vitro LPS-induced TNF-α secretion. Recovery of monocyte function resulted in clearance of sepsis in eight of nine patients. These data suggest that IFN-γ treatment in carefully selected septic patients is a novel therapeutic strategy worth pursuing.

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences.

Abstract: Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was 364 The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of 324, 018, and 015, respectively By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of 005 (*1x2), 013 (*2x2), and 001 (*4x2) Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were 207 (*4), 020 (*3 and *5), 009 (*6), and 001 (*7, *15, and *16) The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found All 41 PMs (70%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T) Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = 006) and IM/PM (mean MR = 014) alleles and between carriers of one (mean MR = 009) and two (mean MR = 003) functional alleles The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment

Substrate specificity of the DnaK chaperone determined by screening cellulose‐bound peptide libraries

Abstract: Hsp70 chaperones assist protein folding by ATP-dependent association with linear peptide segments of a large variety of folding intermediates. The molecular basis for this ability to differentiate between native and non-native conformers was investigated for the DnaK homolog of Escherichia coli. We identified binding sites and the recognition motif in substrates by screening 4360 cellulose-bound peptides scanning the sequences of 37 biologically relevant proteins. DnaK binding sites in protein sequences occurred statistically every 36 residues. In the folded proteins these sites are mostly buried and in the majority found in beta-sheet elements. The binding motif consists of a hydrophobic core of four to five residues enriched particularly in Leu, but also in Ile, Val, Phe and Tyr, and two flanking regions enriched in basic residues. Acidic residues are excluded from the core and disfavored in flanking regions. The energetic contribution of all 20 amino acids for DnaK binding was determined. On the basis of these data an algorithm was established that predicts DnaK binding sites in protein sequences with high accuracy.

P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues

Abstract: When activated, T helper cells differentiate into one of two subsets, Th1 and Th2, characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms involved in protection and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation. Apart from differences in the repertoire of cytokines, no phenotypic attributes are established that distinguish the two subsets. Here we show that Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin. Moreover, only Th1 cells can efficiently enter inflamed sites in Th1-dominated models, such as sensitized skin or arthritic joints, but not in a Th2-dominated allergic response. Immigration of Th1 cells into inflamed skin can be blocked by antibodies against P- and E-selectin. These results provide evidence for adhesion mechanisms to distinguish between the two T helper subsets and mediate their differential trafficking. They indicate that selective recruitment is an additional level of regulation for both effector function profile and character of a local immune response.

Indoor allergen exposure is a risk factor for sensitization during the first three years of life.

Abstract: Background: The purpose of the study was to investigate the influence of environmental allergen exposure on allergic sensitization in infancy and early childhood. Methods: A cohort of 1314 newborns was recruited and followed up prospectively at the ages 12, 24, and 36 months. The levels of major mite (Der p 1 and Der f 1) and cat (Fel d 1) allergens were determined from domestic carpet dust samples by sandwich ELISA. Specific serum IgE antibodies to mite and cat allergens were determined by CAP fluoroimmunoassay (Pharmacia). Logistic regression was used to assess the effects of allergen exposure, age, family history, and cord blood IgE simultaneously on the risk of sensitization. Results: Children, who had been found to be sensitized at least once during the first 3 years of life, were found to be exposed to significantly higher house dust mite (median, 868 ng/gm vs 210 ng/gm; p =0.001) and cat (median, 150 ng/gm vs 64 ng/gm; p =0.011) allergen concentrations in domestic carpet dust compared with the group without sensitization. In homes with low (≤25th percentile) dust concentrations, the risk of sensitization to mite (1.6%) and cat (2.0%) is low, compared with 6.5% for mite and 6.3% for cat if the domestic exposure is above the 75th percentile. The dose-response relationships between allergen levels and sensitization indicate that the increase in sensitization risk at low allergen levels is more pronounced in cat allergy ( p =0.002) than in mite allergy ( p =0.026). In the group with a positive family history, lower mite and cat allergen concentrations are needed to achieve specific sensitization compared with the group with a negative family history. Conclusion: Our data indicate that avoidance measures in the domestic environment aimed at the primary prevention of allergen-driven sensitization should be introduced at the earliest possible stage, if possible during infancy. (J Allergy Clin Immunol 1997;99:763–9.)

EBIC-Guidelines for Management of Severe Head Injury in Adults

Abstract: Guidelines for the management of severe head injury in adults as evolved by the European Brain Injury Consortium are presented and discussed. The importance of preventing and treating secondary insults is emphasized and the principles on which treatment is based are reviewed. Guidelines presented are of a pragmatic nature, based on consensus and expert opinion, covering the treatment from accident site to intensive care unit. Specific aspects pertaining to the conduct of clinical trials in head injury are highlighted. The adopted approach is further discussed in relation to other approaches to the development of guidelines, such as evidence based analysis.

Nuclear localization and increased levels of transcription factor YB-1 in primary human breast cancers are associated with intrinsic MDR1 gene expression.

Abstract: Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.

Spontaneous eye movements during visual imagery reflect the content of the visual scene

Abstract: In nine naive subjects eye movements were recorded while subjects viewed and visualized four irregularly-checkered diagrams. Scanpaths, defined as repetitive sequences of fixations and saccades were found during visual imagery and viewing. Positions of fixations were distributed according to the spatial arrangement of subfeatures in the diagrams. For a particular imagined diagrammatic picture, eye movements were closely correlated with the eye movements recorded while viewing the same picture. Thus eye movements during imagery are not random but reflect the content of the visualized scene. The question is discussed whether scanpath eye movements play a significant functional role in the process of visual imagery.

A Novel Class of RanGTP Binding Proteins

Abstract: The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.

Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension.

Abstract: The human endothelin-1 (ET-1) gene under the control of its natural promoter was transferred into the germline of mice. The transgene was expressed predominantly in the brain, lung, and kidney. Transgene expression was associated with a pathological phenotype manifested by signs such as age-dependent development of renal cysts, interstitial fibrosis of the kidneys, and glomerulosclerosis leading to a progressive decrease in glomerular filtration rate. This pathology developed in spite of only slightly elevated plasma and tissue ET-1 concentrations. Blood pressure was not affected even after the development of an impaired glomerular filtration rate. Therefore, these transgenic lines provide a new blood pressure-independent animal model of ET-1-induced renal pathology leading to renal fibrosis and fatal kidney disease.

Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806

Abstract: Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins. Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases. We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806. Non-hepatotoxic strains of M. aeruginosa lack these genes. Strain PCC 7806 was transformed to chloramphenicol resistance. The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification. This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom-forming cyanobacterium. Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides. The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins. Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and non-toxic strains of M. aeruginosa is the presence of one or more genes coding for microcystin synthetases.

Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis

Abstract: In addition to the RNA polymerases (RNAPs) transcribing the nuclear genes, eukaryotic cells also require RNAPs to transcribe the genes of the mitochondrial genome and, in plants, of the chloroplast genome. The plant Arabidopsis thaliana was found to contain two nuclear genes similar to genes encoding the mitochondrial RNAP from yeast and RNAPs of bacteriophages T7, T3, and SP6. The putative transit peptides of the two polymerases were capable of targeting fusion proteins to mitochondria and chloroplasts, respectively, in vitro. The results indicate that the mitochondrial RNAP in plants is a bacteriophage-type enzyme. A gene duplication event may have generated the second RNAP, which along with the plastid-encoded eubacteria-like RNAP could transcribe the chloroplast genome.

Elevated soluble CD14 receptors and altered cytokines in chronic heart failure.

Abstract: We hypothesized that in patients with chronic heart failure mesenteric venous congestion leads to increased bowel permeability, bacterial translocation, and thereby endotoxin release; the increased endotoxin challenge then causes immune activation with increased soluble CD14 levels and tumor necrosis factor (TNF)-alpha production. Patients with high soluble CD14 levels (indicative of endotoxin-cell interaction) have markedly increased plasma levels of TNF-alpha, soluble TNF receptors 1 and 2, and intracellular adhesion molecule-1, supporting this hypothesis.

Analysis of apoptosis during hair follicle regression (catagen)

Abstract: Keratinocyte apoptosis is a central element in the regulation of hair follicle regression (catagen), yet the exact location and the control of follicular keratinocyte apoptosis remain obscure. To generate an "apoptomap" of the hair follicle, we have studied selected apoptosis-associated parameters in the C57BL/6 mouse model for hair research during normal and pharmacologically manipulated, pathological catagen development. As assessed by terminal deoxynucleotide transferase dUTP fluorescein nick end-labeling (TUNEL) stain, apoptotic cells not only appeared in the regressing proximal follicle epithelium but, surprisingly, were also seen in the central inner root sheath, in the bulge/isthmus region, and in the secondary germ, but never in the dermal papilla. These apoptosis hot spots during catagen development correlated largely with a down-regulation of the Bcl-2/Bax ratio but only poorly with the expression patterns of interleukin-1beta converting enzyme, p55TNFR, and Fas/Apo-1 immunoreactivity. Instead, a higher correlation was found with p75NTR expression. During cyclophosphamide-induced follicle dystrophy and alopecia, massive keratinocyte apoptosis occurred in the entire proximal hair bulb, except in the dermal papilla, despite a strong up-regulation of Bax and p75NTR immunoreactivity. Selected receptors of the tumor necrosis factor/nerve growth factor family and members of the Bcl-2 family may also play a key role in the control of follicular keratinocyte apoptosis in situ.

National rates and regional differences in sensitization to allergens of the standard series : Population-adjusted frequencies of sensitization (PAFS) in 40,000 patients from a multicenter study (IVDK)

Abstract: Sensitization rates to contact allergens vary between centers and are influenced by sex and age. Eliminating the latter 2 factors by standardization of data by age and sex, the present analysis addresses possible differences between centers remaining after elimination of these confounders, and analyzes other factors which might influence rates, e.g., the MOAHL index. Overall standardized rates were well within the range reported in previous studies and may be regarded as representing the rates of the "patch test population" in Central Europe (e.g., nickel sulfate 12.9%, fragrance mix 10.5%, balsam of Peru 7.3%, thimerosal 5.6%). For this analysis, data of those departments which contributed more than 2000 patients, or of those with extreme proportions concerning sex, age and occupational cases were selected. Patients from these 10 departments differed considerably with regard to the items of the MOAHL index and with regard to standardized rates. The items of the MOAHL index proved to be suitable for describing different patch test populations and for explaining some differences between centers. Only 'atopic dermatitis' seems to have little influence on (standardized) rates. Face dermatitis is not yet represented in the MOAHL index, but should be included, together with age > 40 years, in an extended index (acronym: MOAHLFA). Regional allergen exposure (with striking differences between East Germany, West Germany and, to a lesser extent, Austria) seems to have a great influence on the sensitization pattern observed in a department. In addition, sociological factors may influence sensitization rates, which is exemplified by high rates of nickel allergy in a socially defined subgroup. Future studies should focus on these factors, as well as on factors concerning patch test practices and quality control.

Interleukin-15 protects from lethal apoptosis in vivo.

Abstract: Interleukin-15 shares many biological activities with IL-2 and signals through the IL-2 receptor β and γ chains1–3. However, IL-15 and IL-2 differ in their controls of expression and secretion, their range of target cells and their functional activities3–7. These dissimilarities may include differential effects on apoptosis. For example, IL-2 induces or inhibits T-cell apoptosis in vitro, depending on T-cell activation8, whereas IL-15 inhibits cytokine deprivation-induced apoptosis in activated T cells9. Studying whether and how IL-15 modulates distinct apoptosis pathways10–12, we show here that apoptosis induced by anti-Fas, anti-CD3, dexamethasone, and/or anti-IgM in activated human T and B cells in vitro is inhibited by IL-15 in a manner dependent on RNA synthesis. In vivo, anti-Fas-induced lethal multisystem apoptosis in mice is suppressed by a novel IL-15–lgG2b fusion protein. Only IL-15, but not IL-2, completely protected from lethal hepatic failure. Thus, IL-15 is a potent, general inhibitor of apoptosis in vitro and in vivo with intriguing therapeutic potential.

Effects of Magnetic Fluid Hyperthermia (MFH) on C3H mammary carcinoma in vivo

Abstract: Magnetic fluids (MF) have a potential for hyperthermia due to their good power absorption capabilities. Recent in vitro experiments with the so-called 'Magnetic Fluid Hyperthermia (MFH)' have shown that human tumour cells are homogeneously inactivated after AC magnetic field excitation of extracellular MF. The aim of the present study was the evaluation of a high dose MFH on intramuscularly implanted mammary carcinoma of the mouse. The tumours originated from initial in vivo passages of a spontaneous parent tumour. Because of larger variations of tumour growth in this rather primary model, logistic regression of non-averaged volumes was performed for each treatment modality. All growing tumours were randomized 30 days after transplantation (day of treatment) with an overall size distribution between 12M00mm3. An intratumoural steady state temperature of 47 f 1.O"C was maintained for 30 minutes with whole-body AC magnetic fields of 6-12.5 kA/m at 520 kHz. The magnetic fluid was #P6, which is a high biocompatible dextran magnetite. #P6 was given intratumourally (1.5 x lo-' mg ferrite/mm3) 20-30 minutes before excitation and was combined with magnetic targeting (50mT), which yielded a 2.5-fold enhancement of the intratumoural iron concentration. Histological examinations of tumour tissue after intralesional ferrofluid administration alone indicated deep infiltration of the fluid into the carcinoma tissue, but no evidence of tissue damage as compared with untreated controls. In contrast, widespread tumour necrosis was observed after MFH. After application of either dextran or ferrofluid alone (no difference, p = 0.665), tumour growth was slightly delayed in comparison with untreated controls 0) < 0.001). In contrast to the good fit of the controls (R = 0.924.87), tumour growth after MFH was much more heterogeneous; some tumours showed no evidence for regrowth at 50 days whereas others had grown quite readily. This most probably reflected the critical problem of homogeneity of the intratumoural MF distribution, which was also confirmed qualitatively by Magnetic Resonance Imaging (MRI), heterogeneous pigmentation of MFH treated tumours, and up to 1°C differences between temperature probes in the same tumour during AC magnetic field application. However, a quantitative comparison between intratumoural MF-heterogeneity and tumour response could not be performed in this study. Despite these current limitations, the regression analysis of the MFH data yielded a smaller tumour volume of about 1000mm3 at 50 days growth time in contrast to all three controls. In conclusion, encouraging results have been obtained, which show, that one single high dose MFH is already able to induce local tumour control in many cases within 30 days after treatment. To overcome the uncertainties of intratumoural MF heterogeneity, advanced intralesional application methods are currently under development.

The H1 detector at HERA

Abstract: General aspects of the H1 detector at the electron-proton storage ring HERA as well as technical descriptions of the magnet, luminosity system, trigger, slow-control, data acquisition and off-line data handling are given. The three major components of the detector, the tracking, calorimeter and muon detectors, will be described in a forthcoming article. The present paper describes the detector that was used from 1992 to the end of 1994. After this a major upgrade of some components was undertaken. Some performance figures from luminosity runs at HERA during 1993 and 1994 are given.

Sensitization to hen's egg at the age of twelve months is predictive for allergic sensitization to common indoor and outdoor allergens at the age of three years

Abstract: Background: Specific predictors for atopic sensitization in early infancy are prerequisites for preventive intervention studies. Objective: To identify predictors of allergic sensitization to common aeroallergens in infancy, 1314 children in five German cities were followed up from birth (1990) to the age of 3 years. Methods: BLOOD samples were taken from cord blood and at follow-up visits at the ages of 1, 2, and 3 years. Total serum IgE and specific IgE antibodies to common food and inhalant allergens were determined. Results: Among our study population, risk factors for sensitization to indoor and/or outdoor allergens at the age of 3 years were a positive family history, the presence of hen's egg–specific IgE antibodies (G0.35 kU/L), and increased log[total IgE] levels at the age of 12 months. Elevated cord blood IgE was not associated with sensitization to inhalant allergens at the age of 3 years. Egg-specific IgE greater than 2 kU/L in combination with a positive family history of atopy was a highly specific (specificity, 99%) and predictive (positive predictive value, 78%) marker for sensitization to inhalant allergens at 3 years of age. Conclusions: Hen's egg–specific IgE at the age of 12 months is a valuable marker for subsequent allergic sensitization to allergens that cause asthma, allergic rhinitis, and atopic dermatitis. (J Allergy Clin Immunol 1997;99:613-7.)

Basic Number Theory

Abstract: The first goal of algebraic number theory is the generalization of the theorem on the unique representation of natural numbers as products of prime numbers to algebraic numbers. Gauss considered the ring \( \mathbb{Z}\left[ {\sqrt {{ - 1}} } \right] \) of all numbers of the form \( a + \sqrt {{ - 1b}} \) with \( a,\;b \in \mathbb{Z} \) and showed that \( \mathbb{Z}\left[ {\sqrt {{ - 1}} } \right] \) is a ring with unique factorization in prime elements (see §2.1). He introduced these numbers for the development of his theory of biquadratic residues. Another motivation for the study of the arithmetic of algebraic numbers comes from the theory of Tiophantine equations. For example, the quadratic form \( f({x_1},\;{x_2}) = x_1^2 - Dx_2^2 \) with \( D \in \mathbb{Z} \), \( \sqrt {{D\; otin \;\mathbb{Z}}} \) can be written in the form \( \left( {{x_1} - \sqrt {{D{x_2}}} } \right)\left( {{x_1} + \sqrt {{D{x_2}}} } \right) \). Hence the question about the representation of integers by f(a 1, a 2) with \( {a_1},\;{a_2} \in \mathbb{Z} \) can be reformulated as a question of factorization of algebraic numbers of the form \( {a_1} + \sqrt {{D{a_2}}} \). These numbers form a module in the field \( \mathbb{Q}(\sqrt {D} ) \).