[Where is acupuncture going? Letter no. 2].
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Acupuncture↗
Biomedical subjects
H Heine
Publications and source records attributed to H Heine.
In vitro-generated viral double-stranded RNA in contrast to polyinosinic:polycytidylic acid induces interferon-alpha in human plasmacytoid dendritic cells.
Double-stranded RNA (dsRNA) arises in the cytoplasm during viral replication and was shown to participate in the interferon (IFN)-alpha induction process. Besides the intracellular recognition, released dsRNA from dying, infected cells can function as a pathogen-associated molecular pattern (PAMP) for the innate immune system. In the present study, in vitro-generated dsRNA fragments of genomic sequences of Newcastle disease virus were used to induce IFN-alpha release in human peripheral blood mononuclear cells (PBMC), in immature myeloid dendritic cells (mDC) and in immature plasmacytoid DC (pDC). The extracellular administration of dsRNA fragments but not the application of the corresponding single-stranded RNA (ssRNA) strands led to an IFN-alpha production in PBMC. The synthetic dsRNA analogue polyinosinic acid : polycytidylic acid [Poly(I : C)] could only stimulate IFN-alpha production in enriched mDC but not in pDC. In contrast, dsRNA fragments induced IFN-alpha only in pDC. Complexation of dsRNA fragments with transfection reagents increased the efficiency of IFN-alpha induction and commuted ssRNA molecules into IFN-alpha inducers. However, stimulation of in vitro-generated murine Toll-like receptor 7 (TLR7) knockout DC and human TLR-transfected HEK293 cells with dsRNA fragments gave no evidences for the involvement of pDC-specific TLR7 or TLR9 in the observed IFN-alpha induction.
Animals↗
[Stress processing in medicine -- concerning the articles in DMV 3/2005].
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Adaptation, Physiological↗
Structural polymorphism and endotoxic activity of synthetic phospholipid-like amphiphiles.
The physicochemical characteristics and in vitro biological activity of various synthetic hexaacyl phospholipid dimers were compared with the respective behavior of bacterial endotoxins (lipopolysaccharide, LPS). The structural variations of the synthetic amphiphiles include different stereochemical (R,S) configurations about their ester- and amide-linkages for the acyl chains and differences in the length of the serine backbone spacer. The temperature of the gel to liquid crystalline phase transition of the acyl chains (T(c)) lies between 10 and 15 degrees C for the compounds with the shortest backbone and decreases rapidly for the compounds with longer backbones. The phase transition enthalpies (8-16 kJ x mol(-1)) are considerably lower than those of lipid A from hexaacyl endotoxins (28-35 kJ x mol(-1)). In contrast, the dependence of T(c) on Mg(2+) and water content shows a behavior typical for endotoxins: a significant increase with increasing Mg(2+) and decreasing water concentrations. The aggregate structure is sensitively dependent not only on the length of the backbone spacer but also on the different stereochemical variations. It can be directly correlated with the biological activity of the compounds. Thus, as with natural lipid A, the capacity to induce cytokine production in mononuclear cells is directly related to the affinity to form nonlamellar cubic or inverted hexagonal H(II) aggregate structures. Together with the data on the transport and intercalation of the dimers into phospholipid liposomes mediated by the lipopolysaccharide-binding protein (LBP), our conformational concept of endotoxicity and cell activation can be applied to these non-LPS structures: endotoxically active compounds incorporate into membranes of immune cells and cause conformational changes at the site of signaling proteins such as Toll-like receptors or K(+)-channels due to their conical molecular shape.
Animals↗
[Hessian Natural Medicine Award 2003].
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Awards and Prizes↗
Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling.
Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary--K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-alpha or interleukin (IL)-1 beta. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2(C95Y), functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4.
Animals↗
Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts.
The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.
Antigens, CD↗
The biology of endotoxin.
Endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of Gram-negative bacteria and has profound immunostimulatory and inflammatory capacity. The septic shock syndrome caused by endotoxin still has an unacceptably high mortality rate and, owing to increasing numbers of resistant strains, remains an ongoing threat throughout the world. However, the past years have provided new insights especially into the receptors of the innate immune system that are involved into the recognition of LPS and the initial signal transduction pathways that are engaged after the primary recognition on the cell surface. The knowledge about the molecular basis for the responses to endotoxin may eventually lead to the development of new drugs to fight the fatal effects of bacterial infections.
Animals↗
Decay-accelerating factor (DAF/CD55) is a functional active element of the LPS receptor complex.
Previously, we identified an 80 kDa membrane protein (LMP80) that is capable of binding to LPS and lipid A in the presence of LBP and sCD14. LMP80 could also be detected after immuno-coprecipitation of cell membranes with LPS and lipid A, indicating a physical contact of LMP80 and LPS/lipid A. Further analysis and peptide sequencing revealed that LMP80 is identical to CD55 (decay accelerating factor, DAF), a regulatory molecule of the complement cascade. Transfection of LPS-hyporesponsive Chinese hamster ovary (CHO) cells with human CD55 resulted in the translocation of NF-B upon stimulation with LPS or lipid A. Our results demonstrate a new functional role of CD55 as a molecule able to mediate LPS-induced activation of cells that may be part of a multimeric LPS receptor complex.
Animals↗
Bacteraemia and antibiotic resistance of its pathogens reported in England and Wales between 1990 and 1998: trend analysis.
OBJECTIVES: Determination of causes, trends, and antibiotic resistance in reports of bacterial pathogens isolated from blood in England and Wales from 1990 to 1998. DESIGN: Description of bacterial isolates from blood, judged to be clinically significant by microbiology staff, reported to the Communicable Disease Surveillance Centre. SETTING: Microbiology laboratories in England and Wales. SUBJECTS: Patients yielding clinically significant isolates from blood. MAIN OUTCOME MEASURES: Frequency and Poisson regression analyses for trend of reported causes of bacteraemia and proportions of antibiotic resistant isolates. RESULTS: There was an upward trend in total numbers of reports of bacteraemia. The five most cited organisms accounted for over 60% of reports each year. There was a substantial increase in the proportion of reports of Staphylococcus aureus resistant to methicillin, Streptococcus pneumoniae resistance to penicillin and erythromycin, and Enterococcus faecalis and Enterococcus faecium resistance to vancomycin. No increase was seen in resistance of Escherichia coli to gentamicin. CONCLUSIONS: Reports from laboratories provide valuable information on trends and antibiotic resistance in bacteraemia and show a worrying increase in resistance to important antibiotics.
Bacteremia↗
Immunoregulation via 'bystander suppression' needs minute amounts of substances--a basis for homeopathic therapy?
One of the main characteristics of homeopathic drugs is the low concentration of substances they contain. In most discussions this serves as the predominant argument against homeopathic treatments. The small amount of ingredients is in most instances considered not to be able to induce significant changes in classical pharmacological models. A few years ago researchers at the Harvard Medical School in Boston observed that the auto-reactivity ofT-cells is managed by the immune system in at least two different ways that obviously were dependent upon the concentration of the antigen they encounter: If they see high concentrations of a self-antigen they are deleted (killed), but when given low doses they undergo a special kind of active inhibition (called 'bystander suppression'). We feel that this type of regulation induced by very low substance concentrations could serve as a model to explain the way in which at least some homeopathic pharmaceuticals mediate their therapeutic effects.
Homeopathy↗
Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide.
Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.
Animals↗
Bacterial lipopolysaccharide induces expression of the stress response genes hop and H411.
CD14-transfected Chinese hamster ovary K1 fibroblasts (CHO/CD14) respond to lipopolysaccharide (LPS) by metabolizing arachidonic acid and with translocation of NF-kappaB to the nucleus. Although previous experiments failed to identify the production of tumor necrosis factor-alpha and interleukin (IL)-1beta by CHO/CD14 cells, LPS did induce the expression of IL-6 mRNA and the subsequent release of the IL-6 protein. To identify additional LPS-inducible genes, a cDNA library derived from LPS-stimulated CHO/CD14 cells was screened by subtractive hybridization. Fourteen genes were found to be expressed differentially, and two were analyzed in detail: hop (Hsp70/Hsp90-organizing protein), which is the hamster homologue of the stress-inducible yeast gene, STI1, and clone H411, which encodes a novel LPS-inducible growth factor. In response to LPS, the expression of Hop mRNA was also increased in both the murine macrophage cell line, RAW 264.7, as well as in primary hamster macrophages. This suggested that the up-regulation of Hop expression is part of the macrophage stress response to LPS. Clone H411 encodes a protein in the epidermal growth factor-like repeat protein family. Overexpression of H411 cDNA in the RAW 264.7 macrophage cell line promoted an increased growth rate, suggesting that expression of H411 is part of the proliferative cell response to LPS. Both Hop and H411 represent novel gene products not previously recognized as part of the complex biological response to endotoxin.
Amino Acid Sequence↗
Cutting edge: cells that carry A null allele for toll-like receptor 2 are capable of responding to endotoxin.
Toll-like receptor (TLR) 2 and TLR4 have been implicated in the responses of cells to LPS (endotoxin). CD14-transfected Chinese hamster ovary (CHO)-K1 fibroblasts (CHO/CD14) are exquisitely sensitive to endotoxin. Sequence analysis of CHO-TLR2, compared with human and mouse TLR2, revealed a single base pair deletion. This frameshift mutation resulted in an alternative stop codon, encoding a protein devoid of transmembrane and intracellular domains. CHO-TLR2 cDNA failed to enable LPS signaling upon transient transfection into human epithelial kidney 293 cells. Site-directed mutagenesis of CHO-TLR2 enabled expression of a presumed full-length hamster TLR2 that conferred LPS responsiveness in human epithelial kidney 293 cells. Genomic TLR2 DNA from primary hamster macrophages also contained the frameshift mutation found in CHO fibroblasts. Nevertheless, hamster peritoneal macrophages were found to respond normally to LPS, as evidenced by the induction of cytokines. These results imply that expression of TLR2 is sufficient but not essential for mammalian responses to endotoxin.
Alleles↗
Lipopolysaccharide recognition, CD14, and lipopolysaccharide receptors.
The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival. To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria. This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide.
Animals↗
Collagen cross-links in fibromyalgia syndrome.
OBJECTIVE: The acceptance of fibromyalgia as a disease entity and its definitive diagnosis have been hampered by a dearth of knowledge concerning the underlying pathophysiology of this disease and the lack of specific biochemical markers applicable to its diagnosis. To determine whether abnormal collagen metabolism is a characteristic of fibromyalgia, we have analyzed collagen metabolites in the urine and serum of patients with fibromyalgia. METHODS: The diagnosis of fibromyalgia was made according to the American College of Rheumatology criteria. Urine and serum were collected under standardized conditions from 39 patients and 55 age- and sex-matched controls. Pyridinoline (Pyd) and deoxypyridinoline (Dpyd), which represent products of lysyl oxidase-mediated cross-linking in collagen and are indicators of connective tissue and bone degradation, respectively, were analyzed by ion-paired and gradient HPLC method with fluorescence detection (HPLC). Levels of hydroxypyroline (Hyp), a collagen turnover marker, were also measured. The findings were related to creatinine levels and the Pyd/Dpyd ratio determined. RESULTS: The Pyd/Dpyd ratios in the urine and serum and the Hyp in the urine were significantly lower in patients with fibromyalgia than in healthy controls. CONCLUSION: Decreased levels of collagen cross-linking may contribute to remodeling of the extracellular matrix and collagen deposition around the nerve fibers in fibromyalgia and contribute to the lower pain threshold at the tender points. Analysis of altered collagen metabolism either by histologic examination on biopsy or, preferably, by HPLC analysis of collagen metabolites in urine or serum may aid to understand more about the pathogenesis of fibromyalgia.
Amino Acids↗
ADP-ribosylation: role in LPS-induced phosphorylation of two cytosolic proteins (p36/38) in monocytes.
Human monocytes respond to LPS by releasing proinflammatory cytokines such as TNF-alpha, IL-1 and IL-6. Here we show that inhibitors of ADP-ribosylation namely nicotinamide and meta-iodobenzylguanidine prevent production of TNF-alpha and IL-6 at the protein and mRNA level. The inhibitors also influence the LPS-induced phosphorylation pattern of cytosolic proteins. They consistently lead to changes of the phosphorylation state of two proteins with an apparent molecular mass of 36 kDa and 38 kDa. The changes are both time and dose dependent. The data suggest that the conditions leading to altered phosphorylation of p36/38 may correlate with conditions initiating and regulating TNF-alpha and IL-6 production.
3-Iodobenzylguanidine↗
Analysis of hybridoma cell culture processes by SDS/gel capillary electrophoresis and matrix-assisted laser desorption ionization-time-of-flight MS.
SDS/gel capillary electrophoresis (SDS/gel CE) and matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS) were employed to analyse changes in the culture broth during batch and continuous cultivation of hybridoma cells. The stability of IgG was analysed by SDS/gel CE and capillary zone electrophoresis (CZE). The results obtained by the new analytical procedures reflect the changes in the cultivation conditions very well, indicating that these tools can be used to follow animal cell culture processes. A new technique to collect fractions of very small volumes during the CZE separation is described, and the successful off-line coupling of CZE and MALDI-TOF-MS for the analysis of biotechnological processes is demonstrated.
Animals↗