Type IV allergy to natural hirudin confirmed by in vitro stimulation with recombinant hirudin.
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Antithrombins↗
Biomedical subjects
R Kaufmann
Publications and source records attributed to R Kaufmann.
Transcription of melanogenesis enzymes in melanocytes: dependence upon culture conditions and co-cultivation with keratinocytes.
Eumelanogenesis of human skin melanocytes requires at least three enzymes: tyrosinase, TRP 1, and TRP 2. The regulation of these enzymes on transcriptional level was detected in a semiquantitative attempt. The total RNA of melanocytes was reverse-transcripted and followed by a PCR with degenerated primers for all three enzymes. The amplification products were related to each other densitometrically. We examined five different culture conditions: 1) melanocytes in a popular phorbolester containing F-10-medium, 2) melanocytes in a co-culture medium with EGF, 3) melanocytes in a co-culture medium with high calcium, 4) melanocytes co-cultured with keratinocytes in EGF containing co-culture medium, and 5) melanocytes co-cultured with keratinocytes in co-culture medium with high calcium. Melanocytes cultured in phorbolester containing F-10-medium featured transcripts of tyrosinase, TRP 1, and TRP 2 in the ratio 45:45:10. The same results were obtained for melanocytes co-cultured with keratinocytes under the two different culture conditions. In melanocytes cultured alone in co-culture media only TRP 1-transcripts were present. It is likely that under co-culture conditions a keratinocyte-derived factor supports the transcription of all three enzymes. For melanocytes in the phorbolester-containing melanocyte medium a proteinkinase C dependent regulation of transcription seems possible.
Coculture Techniques↗
A model of genital Chlamydia trachomatis infection using human xenografts in severe combined immunodeficiency mice.
We developed a new model of human genital Chlamydia trachomatis infection in order to characterize the pathogen-host relationship in a clinically relevant system using a human strain of C. trachomatis instead of the commonly employed mouse biovar (MoPn). Human endometrial tissue was xenografted into the skin of mice homozygous for the mutation severe combined immunodeficiency and inoculated with C. trachomatis serovar K. C. trachomatis efficiently infected the endometrium as shown by cell culture and immunofluorescence microscopy and persisted for more than 6 weeks. Chlamydial inclusions detected by direct immunofluorescence and electron microscopy appeared to be smaller than those produced by in vitro cell culture-grown chlamydiae. A pattern of localized mild infection prevailed, and infiltrative uncontrolled spread of chlamydiae was observed in only 1 of 10 infected grafts. This might correspond to the well-known tendency of the agent to cause asymptomatic infections. This model allows the study of a human genital infection resembling the clinical situation and offers the possibility to better characterize the host-parasite relationship with respect to pathogenicity and therapy.
Animals↗
Clinical and pathophysiological aspects of hydroxyethyl starch-induced pruritus: evaluation of 96 cases.
BACKGROUND: It has been recently recognized that long-term infusions of hydroxyethyl starch (HES) as a substitute of human plasma may lead to deposits within the human skin and clinically induces severe pruritus in approximately one-third of the patients treated. OBJECTIVE: To analyze the clinical features and pathophysiology of HES-induced itching. METHODS: Clinical data of 86 patients were analyzed retrospectively. Furthermore, 10 patients were followed prospectively, in whom we also performed routine laboratory tests, skin biopsies, allergological investigations (skin prick, basophil degranulation test), and substance P measurements in stimulated macrophages. RESULTS: Special features of HES-induced pruritus included long latency of onset and persistence. Noteworthy, itching is not always generalized. 30% of patients present with localized pruritus. Symptoms were not relieved by antihistaminics. Accordingly, the basophil degranulation test after HES exposure was negative. Also, substance P release from macrophages was not increased following HES stimulation. Deposits were found mainly in macrophages and endothelial cells. CONCLUSIONS: Our findings indicate that pruritus in HES-infused patients is most likely not triggered by pruritogenic mediators. We support data from the recent literature, suggesting a direct stimulation of cutaneous nerves by HES deposits. In patients suffering from long-standing itching it seems important to consider previous HES exposure as a possible cause not only in generalized pruritus but also in localized symptoms.
Biopsy, Needle↗
Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.
A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.
Affinity Labels↗
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry: a novel analytical tool in molecular biology and biotechnology.
Matrix-assisted laser desorption ionization (MALDI) is a rather recent 'soft ionization' technique which produces quasimolecular ions of large organic molecules of up to several 100 kDa molecular mass. In combination with time-of-flight (TOF) mass spectrometry it has rapidly evolved as a valuable tool for the detection and characterization of biopolymers such as peptides, proteins, oligosaccharides and -nucleotides especially in mixtures and crude samples. MALDI-MS is remarkably tolerant towards common contaminants such as salts and buffers, requires sample loads in the subpicomolar range and features a mass accuracy (and precision) of 0.1-0.01%. Structural information on analyte molecules can be obtained by exploiting metastable post source decay (PSD) processes which allow extraction of sequence or substituent information on individual peptides contained, e.g., in an enzymatic digest. Thus, MALDI provides for an easy and straight forward elucidation of posttranslational modifications. The forseeable merging of MALDI with blotting and in situ degradation techniques give MALDI-MS a key role in future laboratory practice of biotechnology and molecular biology. The actual state-of-the-art is described, and selected examples are given with special emphasis on the aspects of analytical routine work.
Animals↗
Occurrence of aluminium in chloride cells of Perla marginata (Plecoptera) after exposure to low pH and elevated aluminum concentration.
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Aluminum↗
Development and characterization of a wettable surface modified aromatic polyethersulphone using glow discharge induced HEMA-graft polymerisation.
The aromatic polyethersulphone (PES) is a well known polymer for the preparation of membranes with excellent thermal stability and chemical resistance. The disadvantage of PES-membranes is their hydrophobic character, which in contact with protein containing solutions leads to high protein adsorption and as a consequence to deterioration of membrane properties. In this report the surface modification of PES by means of glow discharge induced grafting of 2-hydroxyethyl methacrylate (HEMA) is described. Graft polymerisation creates a largely wettable layer of poly(2-hydroxyethyl methacrylate) (PHEMA) on the surface of PES. This has been shown by contact angle measurements using the Wilhelmy plate method. Chemical characterization is carried out by means of X-ray photoelectron spectroscopy (XPS) and infrared spectroscopy/attentuated total reflection (FTIR-ATR). The influence of storage conditions on the surface properties of modified PES samples has been investigated after storage in vacuum, water, and air.
Journal Article↗
CCKB receptor activation results in increased [3H]thymidine incorporation in rat glioma C6 cells.
Cholecystokinin (CCK) is known to stimulate cell proliferation but involvement of CCKB type receptors has not been exactly demonstrated so far. We examined the effect of CCK-8S and two receptor agonists on rat glioma C6 cells when using different CCKB receptor agonists and antagonists. Both CCK-8S and CCKB receptor agonists BC 264 and Suc-Trp-N(Me)Nle-Asp-Phe-NH2 stimulate [3H]thymidine incorporation. These effects were inhibited by CCKB receptor antagonist L-365,260 over 100-fold more effectively than it was seen by using CCKA receptor antagonist L-364,718. The data indicate that CCKB receptor agonists are potent stimulants of rat glioma C6 cell DNA synthesis suggesting that CCKB receptor activation is involved in cell proliferation within the central nervous system.
Amino Acid Sequence↗
Cholecystokinin induced signaling in rat glioma C6 cells.
We investigated the effect of sulphated cholecystokinin octapeptide (CCK-8S) on free intracellular calcium concentration, phosphatidylinositol metabolism, and protein phosphorylation in C6 cells, a rat glioma cell line which was shown to express CCKB type receptors. Increase in [Ca2+]i by both influx across the cell membrane and release from internal stores was demonstrated by utilizing a laser confocal imaging system. Because CCK-8S produced a transient elevation of inositol triphosphate level participation of InsP3 in calcium signaling in C6 cells is very likely. Protein kinase C seems to be involved in CCK-8S induced signaling in rat glioma C6 cells as demonstrated by using in vivo phosphorylation experiments.
Animals↗
Effects of guanyl nucleotides on CCKB receptor binding in brain tissue and continuous cell lines: a comparative study.
The effects of non-hydrolyzable guanyl nucleotide analogue GTP-gamma S on CCKB receptor binding in human and guinea-pig cortex, Jurkat T-cells, rat pituitary GH3 cells, rat glioma C6 cells and human small cell lung cancer NCI-H69 cells were investigated by using [3H]CCK-8S saturation and competition binding studies. GTP-gamma S caused inhibition of specific [3H]CCK-8S binding in a concentration dependent manner with a plateau at 10-25 microM. 25 microM GTP-gamma S resulted in a small but significant increase in Kd and IC50 values with amount very similar in all CCKB receptor models tested. However, the maximal number of specific [3H]CCK-8S binding sites (Bmax) was unaffected. Results suggest that CCKB receptors are G-protein coupled in a similar way to human and guinea-pig cortex, Jurkat cells, GH3 cells, C6 cells and NCI-H69 cells.
Animals↗
Megakaryocytes and fibroblasts--interactions as determined in normal human bone marrow specimens.
An in vitro study was performed to investigate possible interactions between megakaryocytes and bone marrow fibroblasts, both obtained from healthy donors. We were able to demonstrate that the proliferation of fibroblasts increased significantly by co-culturing these cells with megakaryocytes for 6 days. Addition of neutralizing antibodies for PDGF and TGF beta 1, caused a significant reduction of fibroblast growth. Inhibition of cell to cell contacts via tissue culture inserts generated a conspicuous impairment of fibroblast proliferation compared with megakaryocyte-fibroblast co-cultures, where contact was allowed. Hence, our findings suggest that a close spatial relationship between megakaryocytes and fibroblasts is needed for the activation of growth in normal human bone marrow. Neighbouring of megakaryocytes and fibroblasts seems to be necessary in order to achieve a certain threshold of local growth factor concentration. Our results are in keeping with the assumption that PDGF and TGF beta 1, are secreted by normal human megakaryocytes in very low concentrations and promote significantly fibroblast proliferation.
Adult↗
Amelanotic lentigo maligna melanoma.
Only five cases of the amelanotic variant of lentigo maligna melanoma of the face have been reported. We describe an additional four patients observed within the past 5 years, suggesting a much higher incidence of this variant than previously suspected.
Aged↗
A subset of macrophages located along the basement membrane ("lining cells") is a characteristic histopathological feature of psoriasis.
In this paper, we describe the immunophenotype and ultrastructure of a new macrophage subpopulation present in inflamed but not in normal human skin. In biopsy material from patients with psoriasis (n = 4), atopic dermatitis (n = 4), and positive patch test reactions (n = 4), spindle-shaped macrophages located along the basement membrane were detected staining positive for CD11c (LeuM5 high, Ki-M1 low), CD68 (Ki-M6 low), and Ki-M8 (low). This subpopulation was most prominent in psoriasis, where a median of 34 macrophages neighbored 100 basal keratinocytes, thus covering about 80% of the dermoepidermal junction. The numbers of atopic and positive patch test reactions were 11 and 13, respectively, accounting for about 20% of the dermoepidermal junction. Transmission electron microscopy revealed an electron-lucent nucleus with dense deposits of chromatin along the nuclear membrane. Interruptions of the integrity of the basement membrane neighboring these cells were also seen. We repeatedly observed parts of basal keratinocytes merging through these openings and macrophages exhibiting cytoplasmic extentions directed toward the porus. Our observations suggest vigorous interactions between macrophages and basal keratinocytes and support the hypothesis that these interactions are an important regulatory event in the pathogenesis of psoriasis.
Antigens, CD↗
Properties of the carcinoma-associated antigen MH 99/KS 1/4 in normal and transformed human keratinocytes: regulation of synthesis, molecular cross-linking and ultrastructural localization.
A 38-kDa cell-surface glycoprotein defined by monoclonal antibody MH 99 is markedly increased in many epithelial tumours. In normal human skin, it is a characteristic marker for germ-cell phenotypic tissues. Although the gene encoding the MH 99 antigen has recently been cloned, and several histological and biochemical studies have been performed, the biological function of this interesting antigen still remains unknown. In the present study, we examined the synthesis of MH 99 in keratinocyte populations showing different in vitro differentiation capacity. Normal keratinocytes, spontaneously immortalized keratinocytes (cell line HaCaT), three SV-40-transformed keratinocyte lines (130, 425, and HaSV), and two squamous cell carcinoma lines (SCL-1 and SCL-2), were compared. Radioimmunoprecipitation revealed the highest levels of synthesis in cell populations with the least differentiation. This was paralleled by an increase of MH 99 synthesis in normal keratinocytes cultured in low concentrations of Ca2+ and by an increase of MH 99 synthesis during subculture of normal keratinocytes. Both phenomena were paralleled by an opposite behaviour of a differentiation marker. Molecular cross-linking and subsequent immunoprecipitation led to a decrease of the MH 99 signal, but an increase of a high molecular weight protein signal was seen. After cleavage of the crosslinker, the MH 99 signal reappeared, whereas the signal of the large protein remained unchanged. Thus, the MH 99 antigen may be associated with a high molecular weight protein on the cell surface, supporting the suggestion of a receptor-like function. Phosphorylation of the molecule could not be detected. Immunoelectron microscopy revealed homogeneous distribution on the cell surface, but cells of the same culture exhibited clear differences in their MH 99 expression. A concept for MH 99 regulation in normal and transformed human keratinocyte populations in vitro is proposed, showing that the synthesis of MH 99 is inversely correlated with cell differentiation. The association with a high molecular weight protein supports the suggestion that the MH 99 antigen interacts with other molecules.
Antibodies, Monoclonal↗
Fuzzy logic and control: principal approach and potential applications in medicine.
During the last few years intelligent machines appeared in nearly all technical areas, such as consumer electronics, robotics, and industrial control systems. There are for example washing machines that work very effectively, need comparably less power than in the past, and have short execution times because they adjust their washing cycles to each set of clothes and change their washing strategies as the clothes become clean. These intelligent systems are based on fuzzy control strategies, i.e., common sense rules are used to describe a system's behavior instead of complex mathematical models. We have applied this new technology to control problems as well as to reasoning problems in biomedical engineering where appropriate mathematical models could not be built due to the complexity of the problem. After a short introduction to the concepts of fuzzy logic two approaches in the field are described: a fuzzy control strategy for the pump rate adjustment of a novel total artificial heart and an intelligent alarm system based on fuzzy inference which supports the anesthetist in monitoring and evaluating the hemodynamic state of a patient undergoing cardiac surgery. These examples indicate the inherent reliability and stability of this technique in the field of complex dynamic systems. Such properties are highly significant especially in medical applications.
Anesthesia↗
Fuzzy control concept for a total artificial heart.
The development of an electromechanically driven total artificial heart (Helmholtz-TAH) was initiated in 1990. Anatomical fitting, biocompatibility, and automatic physiologic adaptation of pump output are the basic requirements that characterize the overall TAH concept. For evaluation of these features, a TAH labtype was developed. It provides most features of the conceptual artificial heart and supports in vitro testing of energy conversion, pump behavior, structural parts, sensors, and control concepts. A fuzzy controller has been implemented for adaptation of the pump rate to body perfusion demand by left pump chamber filling detection. This controller will be an important element of a future extensive TAH control system. The implementation is supported by a professional fuzzy control development tool that allows on-line and real time optimization of control strategies for dynamic processes. The first experiments proved the feasibility and the advantages of this fuzzy control concept. The first in vitro test results are presented.
Fuzzy Logic↗
Influence of cytokines (IL-1 alpha, IL-3, IL-11, GM-CSF) on megakaryocyte-fibroblast interactions in normal human bone marrow.
The evolution of myelofibrosis accompanying chronic myeloproliferative disorders (CMPDs) is often linked with megakaryopoiesis. However, it is not known whether or to what extent megakaryocytes of normal human bone marrow are capable of stimulating fibroblast growth. For this reason, an in vitro study was performed to elucidate possible cytokine-dependent interactions between megakaryocytes and fibroblasts derived from healthy volunteers. Fibroblast growth was significantly promoted by the presence of megakaryocytes and modulated by additional application of various cytokines. While recombinant human (rh) interleukin (IL)-1 alpha had no obvious effect on fibroblast proliferation, a slight increase was detected on adding granulocyte-macrophage colony stimulating factor (rhGM-CSF). Application of rhIL-3 caused a significant increase in the number of fibroblasts. In contrast, administration of rhIL-11 suppressed the megakaryocyte-dependent growth-promoting effect and co-stimulation with rhIL-3 led to a significant decrease of fibroblast number in comparison to rhIL-3-stimulated co-cultures. Inhibition of cell-cell contact in unstimulated, as well as in rhIL-3-stimulated co-cultured led to a conspicuous impairment of fibroblast growth. A similar effect was observed when neutralizing antibodies directed against platelet-derived growth factor (PDGF) and transforming growth factor (TGF)beta 1 were added to rhIL-3-stimulated cultures. Our findings are in keeping with the assumption that interactions between megakaryocytes and fibroblasts involve in cytokine-mediated functional network regulated by factors such as spatial relationship, cytokine stimulation, and low concentrations of mediators, particularly PDGF and TGF beta. In this complex system rhIL-3 seems to play a crucial role in the promotion of these various interrelationships.
Bone Marrow↗