Cooperativity between antibiotics and antiseptics: testing the bactericidal effect.

OBJECTIVE: Treatment with antibiotics together with local application of antiseptics is common in wound care. We investigated the effectiveness of an antiseptic in two variations: octenidine (Oct) and octenidine+ (Oct+ with isotonic glucose addition). METHOD: Using the agar diffusion test with cultures of pathogenic Staphylococcus aureus and the non-pathogenic Bordetella petrii, we compared the effectiveness of octenidine to the classical antiseptics beta-isodona (povidone-iodine; PI), chlorhexidine (Chl) and taurolin (Tau) alone, and in combination with various common antibiotics to uncover cooperativity between antiseptics and antibiotics. RESULTS: We detected strong interactions between antibiotics and antiseptics, that either enhanced or reduced the bactericidal efficiency. Effectiveness was dependent on the type of organism tested. Oct applied together with ineffective antibiotics frequently led to effective growth inhibition of Bordetella petrii. With Staphylococcus aureus we did not find such an effect. To this end, we reason that positively charged Oct may associate with antibiotic compounds via electrostatic interactions and guide it more efficiently to the bacterial cell wall. Interaction with antibiotics sometimes led to sequestration and reduced availability of some antiseptic/antibiotic combinations, but never with Oct. CONCLUSION: These data provide new arguments for decision planning concerning the choice of agent in the treatment of wound infections.

Cell growth and migration under octenidine-antiseptic treatment.

OBJECTIVE: The toxicity of octenidine antiseptics in cultured cells contrasts their good tolerability in tissue. This phenomenon prompted us to examine which cell culture conditions allow survival and proliferation and to investigate a possible modulation of toxicity by the extracellular matrix proteoglycan chondroitin sulfate. METHOD: We tested fibroblasts and MCF7 cells for growth using the MTT test, and assessed wound healing potency with a laceration assay. Expression levels of the genes involved in controlling wound healing were assessed with RT-PCR. RESULTS: A 24 hour exposure to the octenidine-based solution was found incompatible with cell growth. When octenidine solution (0.5-0.5mg/l) was coated on dishes, growth was profoundly reduced after 24 hours, however there was no cytotoxic effect at 0.012 mg/l. Interestingly, when dishes were first coated with chondroitin sulfate the cytotoxicity of octenidine-based solution was modulated. Cell migration was not inhibited by octenidine-based solution treatment (2 minutes; 15 mg/l). No significant changes in gene expression levels in response to the octenidine-based solution treatment were detected. CONCLUSION: In cell culture conditions application of the octenidine-based solution without toxicity can be observed, comparable to the minimal application required to give full bactericidal effect. Alteration of toxicity by interaction with chondroitin sulfate in cell culture suggests a similar function for extraceullar matrix in intact tissue.

Signal transduction and metabolism in chondrocytes is modulated by lactoferrin.

OBJECTIVE: Activation of granulocytes causes a considerable rise in the concentration of lactoferrin (Lf) in synovial fluid (SF). We here investigate consequences thereof on signal transduction and the balance between catabolic and anabolic metabolism in chondrocytes. METHODS: Signal transduction was analysed in cultured chondrocytes by immunodetection of mitogen activated protein kinases (MAPK) and analysis of Smad2 translocation to the nucleus. Expression levels of matrix metalloproteinases (MMPs) and of aggrecan were measured by reverse-transcription-PCR. The proteolytic activity of MMPs was ascertained by zymography. Expression of the low-density-lipoprotein-receptor-related-protein-1 (LRP-1), a Lf receptor for signalling, was assayed by immunohistochemistry in cartilage and in cultured chondrocytes by immunoblotting. RESULTS: We found LRP-1 expressed in dedifferentiated chondrocytes in culture and in cartilage tissue preferentially on the articular surface where it can encounter Lf within SF. Lf stimulated proliferation of chondrocytes, comparable to transforming growth factor-beta1 (TGFbeta1) and activated p38 and the extracellular-signal regulated-kinases 1/2 (ERK1/2) within minutes. Surprisingly, Lf induced nuclear Smad2 translocation, a signal pathway ascribed to TGFbeta receptor activation. Lf significantly increased the levels of catabolic indicators such as MMP1, MMP2, MMP3 and MMP13 and inhibited aggrecan synthesis. CONCLUSION: Lf is a robust regulator of chondrocyte metabolism, comparable to TGFbeta1. The catabolic influence together with the proliferative stimulus indicates a function as an early phase cytokine, enhancing MMPs, necessary for degradation of damaged tissue and stimulating proliferation of chondrocytes, necessary for reconstruction.

Assorted effects of TGFbeta and chondroitinsulfate on p38 and ERK1/2 activation levels in human articular chondrocytes stimulated with LPS.

OBJECTIVES: Inadequate cellular response of chondrocytes to stress frequently terminates in osteoarthritis (OA). Adequate response is fundamentally modulated by concerted cytokine signaling events, directing degradation and synthesis of cartilage on articular surfaces where and whenever necessary. Transforming growth factor (TGF)beta is a prominent mediator in cartilage anabolism, although particular catabolic activities are occasionally reported. Clearly, before the TGFbeta signal gets through to the gene regulatory machinery, cross talk with modulators occurs. METHOD: We tested the hypothesis whether chondroitinsulfate (CS) modulates cell signaling. TGFbeta and/or soluble CS was added to human articular chondrocytes (HACs) and activation of p38 and extracellular signal related kinase (ERK)1/2 was determined by immunoblot analysis. Expression levels of mRNA of matrix metalloproteinase (MMP)-2, -3 and -13 were determined by real-time polymerase chain reaction (PCR). RESULTS: No significant effects were observed unless cells were stimulated with lipopolysaccharide (LPS), invigorating catabolic metabolism in chondrocytes. LPS effects, however, were profoundly modulated by TGFbeta, CS and both applied in combination. Most prominent, the silencing of p38 stress signal by CS was superimposable to that of TGFbeta. Phospho-ERK1/2 levels were raised by TGFbeta three-fold over LPS induced levels. In contrast, CS treatment, alone or combined with TGFbeta, reduced phosphorylation significantly below LPS induced levels. Finally, suppression of LPS induced MMP-13 mRNA levels resulted with CS. CONCLUSION: Soluble CS modulates signaling events in chondrocytes concurrent with MMP-13 down regulation. The effects observed suggest a feedback signaling mechanism cross talking with TGFbeta-signal pathways and may serve an explanation, on the cellular level, for the beneficial effects found in clinical studies with pharmacologic application of CS.

Apolipoprotein E (apoE) uptake and distribution in mammalian cell lines is dependent upon source of apoE and can be monitored in living cells.

As part of investigations of the cellular uptake of apolipoprotein E (apoE) relevant to Alzheimer's disease we have found that different preparations of apoE are handled differently by cells expressing the LDL-receptor. Comparing recombinant, cellular and native apoE, complexed with different preparations of lipid we find that only cellular and native apoE enter a vesicular compartment. Some, but not all of these apoE containing vesicles are lysosomes. In order to further examine the intracellular fate of apoE we demonstrate that apoE-Enhanced green fluorescent protein chimeric protein can be taken up from medium by recipient cells and tracked within these cells for extended periods.

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor.

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.

Apolipoprotein E and complement C3 polymorphism and their role in the response to gemfibrozil and low fat low cholesterol therapy.

Three different allelic variants of apolipoprotein E determine, in concert with other gene products, the levels of plasma lipoproteins. Recently, cleavage products of the complement C3 molecule have also been implicated in determining plasma triacylglycerol concentrations. This study presents data of an ongoing study to dissect the role of the apolipoprotein E gene locus in the response to low fat/low cholesterol diet combined with gemfibrozil treatment. In addition, for the first time, the significance of C3 allelic variants to such hypolipidaemic therapy response was analysed. To this end data from 81 obese hyperlipoproteinaemic patients (Fredrickson type II/A and B and type IV and V) confirmed the usefulness of the combined gemfibrozil/diet treatment and unveiled apolipoprotein E allele group specific therapy responses. The mean changes of lipid properties due to combined treatment was 15% for total cholesterol, 48% for triacylglycerols and 28% for atherogenic index. Division into hyperlipidaemia types according to Fredrickson and subgrouping into E2, E3 and E4 groups (apolipoprotein E2/2 and 2/3, apolipoprotein E3/3 and apolipoprotein E4/2 and 4/3 phenotype groups respectively) exposed pronounced differences from these mean changes, suggesting substantial influence of apolipoprotein E variants on this therapy. We observed triacylglycerol reductions of from 17% in type IIA-apolipoprotein E3 group patients up to 78% in the type IV and V-apolipoprotein E2 group. Thus it might be concluded the apolipoprotein E genotyping aides therapy success prediction. Although, low sample number in some subgroups obscures significance in this pilot study, significant therapy success emerges for the E3 and E4 group in type IV and V hyperlipidaemia and type IIB-apolipoprotein E3 homozygous patients can be predicted to respond better than apolipoprotein E2 carriers. Finally, we present evidence that positive changes of lipid properties are also determined by the "fast" complement C3 allel (C3-F). Patients with complement factor C3-FS pattern respond better to treatment than patients with C3-SS configuration. In summary these data endorse the genotyping of apolipoprotein E alleles to predict maximal success of "fibrate" treatment. In addition they argue strongly for further assessment of the involvement of complement C3 allelic variations in lipid homeostasis.

Removal of lactoferrin from plasma is mediated by binding to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor and transport to endosomes.

LDL receptor related protein (LRP) is a ubiquitously expressed cell surface receptor that binds, at least in vitro, a plethora of ligands among them alpha 2-macroglobulin and lactoferrin (Lf). The function of LRP in internalisation and distribution of ligands within cellular metabolism is still unclear. We here investigated by combined ligand- and immunoblotting the participation of LRP/alpha 2MR and its associated protein (RAP) in receptor mediated endocytosis of Lf into rat liver. We found LRP highly enriched in sucrose density gradient fractions around density 1.10 g/ml, previously characterised as endosomal fractions. RAP was concentrated in distinct fractions around density 1.14 g/ml. This separation of RAP from LRP/alpha 2MR is physiologically meaningful as RAP avidly binds to LRP/alpha 2MR and by that shuts off all ligand binding function. In endosomal fractions we found one single binding protein for 125I-labelled Lf. With a specific anti LRP/alpha 2MR antibody and ligand blotting with 125I-labelled RAP this endosomal Lf binding site was verified to be LRP/alpha 2MR. Endosomes did not bind labelled Lf when prepared from rats that received an intravenous injection of Lf (20 mg per animal) 20 min prior to preparation. Surprisingly we immunodetected Lf in these endosomes at a position around 600 kDa, comigrating with LRP/alpha 2MR. We determined Lf binding to be optimal at pH 5.8, what led us to suggest the existence of a very stable LF-LRP/alpha 2MR complex in endosomes. These data support the idea of effective binding of Lf at pH as found in inflamed tissue environment where Lf is reported to be involved in leukocyte mediated inflammation regulation.

Members of the low density lipoprotein receptor family mediate cell entry of a minor-group common cold virus.

A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the alpha 2-macroglobulin receptor/LDLR-related protein (alpha 2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptor-associated protein (RAP), providing evidence that alpha 2MR/LRP also acts as a minor group HRV receptor.

Hypolipidemic activity of HOE-402 is mediated by stimulation of the LDL receptor pathway.

HOE-402 (4-amino-2-[4,4-dimethyl-2-oxo-1-imidazolidinyl]-pyrimidine-5-N- [trifluoromethylphenyl]-carboxamide-monohydrochloride) has been shown to exhibit hypolipidemic action in heterozygous Watanabe heritable hyperlipidemic rabbits. In all animals, elevated cholesterol levels were reduced to normal (from 3.0 to 1.5 mmol/L) after 3 weeks of HOE-402 treatment. This was due entirely to reduction of low density lipoprotein (LDL) cholesterol and was paralleled by accelerated removal of plasma 125I-LDL. This reduction of LDL levels was not found in homozygous LDL receptor-defective animals, emphasizing the necessity of a functional LDL receptor system for the hypolipidemic action. The effect of HOE-402 on LDL receptor activity in the cultured hepatoma cell line HepG2 was also determined. When cells were incubated with plasma from treated animals (containing cholesterol 1.5 mmol/L and HOE-402 80 ng/mL), high-affinity cell-surface binding sites for LDL were induced more than threefold, as shown by Scatchard analysis of cell-surface binding data. Induction of the LDL receptor was detectable after 6 hours and was 300% after 18 hours. This induction was specific for LDL, as 125I-transferrin and [59Fe]transferrin were internalized normally in HOE-402-treated cells. The increase of LDL receptor protein was related to induced LDL receptor mRNA levels (400%), as shown by quantification of Northern blotting experiments. These findings suggest that HOE-402 mediated its hypolipidemic action mainly via the LDL receptor pathway. It enhanced mRNA levels for LDL receptor, hence increasing its synthesis, which subsequently resulted in reduced plasma LDL levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactoferrin specifically inhibits endocytosis of chylomicron remnants but not alpha-macroglobulin.

Our recently found nonlipoprotein inhibitor of chylomicron remnant uptake, lactoferrin, has been investigated in vivo and in vitro. Lipoprotein lipase extracted triglycerides from chylomicrons, doubly labeled with [3H]retinol/[14C]oleate, in the presence of lactoferrin normally. The subsequent uptake of remnants into liver was retarded considerably. In the intact rat, chylomicron remnants (CRs), predominantly labeled in the apoB48 moiety by 125I, were excluded from the hepatic endosomal compartment in the presence of lactoferrin as shown in subcellular fractionation studies of rat livers. In tissue culture, internalization of [125I]chylomicron remnants was inhibited in the presence of 14 pM lactoferrin by 70%. Upon removal of lactoferrin, internalization was rapidly restored. Protease digestion eliminated the inhibitory effect completely. Modification of arginine residues with cyclohexanedione reversibly removed the inhibitory potency of lactoferrin. We located by molecular modeling an alpha-helical segment in lactoferrin on the exposed surface of the molecule containing the sequence Arg-X-X-Arg-Lys-X-Arg, which resembles the receptor recognition structure in apolipoprotein E (apoE). This firmly established ligand correspondence with apoE, the candidate ligand for CR recognition by the receptor. Finally, the postulated second function of low density lipoprotein receptor-related protein, uptake of alpha-2-macroglobulin (alpha 2M) was found to be distinct from lipoprotein binding, since lactoferrin inhibited CR but not alpha 2M internalization. In addition, CR uptake was not affected by alpha 2M. We conclude that if a bifunctional receptor were to operate, its diverse functions were exerted by independently operating substructures. The results of our in vivo and cell culture experiments are, however, entirely compatible with the existence of two receptors as well.

Characteristics of chylomicron remnant uptake into rat liver.

We have investigated uptake of 125I-labeled chylomicron remnants into livers of rats in the presence of lactoferrin. This glycoprotein possesses a cluster of four arginines at the N-terminus similar to the arginine rich binding sequence of apoprotein E (apoE) to the LDL-receptor. We found that this protein inhibits uptake of 125I-chylomicron remnant radioactivity by 50% when measured as accumulation of radioactivity into the intact organ, and even more pronounced, over 75%, when measured as uptake into an endosomal fraction prepared therefrom. Provided that the arginine rich sequence is responsible for the inhibition, a similarity in the characteristics of binding of apoE to the LDL (low density lipoprotein)- and chylomicron remnant-receptor is likely. Second, transferrin having sequence homologies with lactoferrin, but lacking the arginine cluster does not interfere with chylomicron remnant uptake. Third, lactoferrin does not inhibit the uptake of chylomicron remnants by the spleen, which is most likely mediated through scavenger cells by a mechanism different from the chylomicron remnant uptake system of the liver. We hypothesize from this that lactoferrin specifically interferes with the physiologically relevant chylomicron remnant uptake system of the liver. Investigation of the mechanism of this inhibition will provide information about the physical characteristics of the remnant receptor system.

Role of receptor-independent low density lipoprotein transport in the maintenance of tissue cholesterol balance in the normal and WHHL rabbit.

These studies were undertaken to determine the role of receptor-independent low density lipoprotein (LDL) transport in cholesterol balance across individual tissues and the whole animal. Homologous LDL, which measures total LDL transport, and methylated heterologous LDL, which measures receptor-independent LDL uptake, were cleared from the plasma at very different rates in the NZ control rabbit (3,900 and 1,010 microliter/hr per kg, respectively) whereas in the WHHL rabbit both preparations were cleared at essentially the same rate (approximately 1,070 microliter/hr per kg). Receptor-independent LDL clearance was detected in all tissues of the NZ control rabbit and these varied from 32 (spleen) to less than 0.5 (skeletal muscle) microliter/hr per g. In contrast, receptor-dependent LDL uptake was found in only about half of these same organs. In the WHHL rabbit, the rates of receptor-independent LDL transport were the same as in the NZ control rabbit, but no receptor-dependent uptake was detected. Using these clearance values it was calculated that in the control rabbit nearly 70% of LDL-cholesterol was removed from the plasma by the liver and 89% of this was receptor-mediated. With loss of receptor activity, however, the burden of LDL degradation was shifted away from the liver so that approximately 70% of LDL-cholesterol uptake took place in the extra-hepatic tissues of the WHHL rabbit. Thus, in the normal animal, the primary function of receptor-dependent LDL transport is to promote the rapid uptake and disposal of plasma LDL by the liver. In the absence of such receptor activity, cholesterol balance across most individual organs and the whole animal remains essentially normal and is mediated by the receptor-independent process. Because of the much lower absolute clearance rates manifested by this transport mechanism, however, substantial and predictable elevations in the circulating plasma LDL-cholesterol levels are required to maintain this balance.

Imaging of hepatic low density lipoprotein receptors by radionuclide scintiscanning in vivo.

The low density lipoprotein (LDL) receptor mediates the cellular uptake of plasma lipoproteins that are derived from very low density lipoproteins (VLDL). Most of the functional LDL receptors in the body are located in the liver. Here, we describe a radionuclide scintiscanning technique that permits the measurement of LDL receptors in the livers of intact rabbits. 123I-labeled VLDL were administered intravenously, and scintigraphic images of the liver and heart were obtained at intervals thereafter. In seven normal rabbits, radioactivity in the liver increased progressively between 1 and 20 min after injection, while radioactivity in the heart (reflecting that in plasma) decreased concomitantly. In Watanabe-heritable hyperlipidemic rabbits, which lack LDL receptors on a genetic basis, there was little uptake of 123I-labeled VLDL into the liver and little decrease in cardiac radioactivity during this interval. These findings demonstrate that the LDL receptor is necessary for the hepatic uptake of VLDL-derived lipoproteins in the rabbit. Two conditions that diminish hepatic LDL receptor activity, cholesterol-feeding and prolonged fasting, also reduced the uptake of 123I-labeled VLDL in the liver as measured by scintiscanning. The data suggest that radionuclide scintiscanning can be used as a noninvasive method to quantify the number of LDL receptors expressed in the liver in vivo.

Use of monoclonal anti-receptor antibodies to probe the expression of the low density lipoprotein receptor in tissues of normal and Watanabe heritable hyperlipidemic rabbits.

Watanabe Heritable Hyperlipidemic (WHHL) rabbits, like humans with familial hypercholesterolemia, have a genetic defect in the low density lipoprotein (LDL) receptor. WHHL fibroblasts produce a low molecular weight precursor form of the receptor that is not glycosylated normally and is not transported to the cell surface at a normal rate. In the current studies, we have used a monoclonal antibody that reacts with the rabbit LDL receptor to extend these findings to intact rabbits. We have made the following observations: (a) In normal rabbits the liver and adrenal glands synthesize high molecular weight mature LDL receptors like those in fibroblasts. (b) In WHHL rabbits the adrenals express only the low molecular weight receptor precursor, and the liver expresses no detectable receptors. (c) When injected intravenously, the radioiodinated anti-LDL receptor monoclonal antibody is cleared from plasma 6-10-fold faster in normal than in WHHL rabbits, with specific uptake demonstrable in livers and adrenals of normal but not WHHL rabbits. The latter finding raises the general possibility that the total number of cell surface receptors expressed by an animal or human in vivo can be estimated by measuring the rate of clearance of an intravenously injected monoclonal antibody directed against the receptor of interest.