Analysis of amphotericin B-induced cell signaling with chemical inhibitors of signaling molecules

Although amphotericin B (AmB) is a major polyene antibiotic against invasive fungal infection, administration to patients sometimes causes inflammatory side effects, which limits the usage of the antibiotic. We studied the intracellular signaling that was induced by AmB. p65 (RelA) of nuclear factor‐κB (NF‐κB), a well‐known signaling molecule as an inducer of proinflammatory cytokines, was phosphorylated by AmB in RAW264.7 cells, a monocyte‐like cell line. Among chemical inhibitors of signaling molecules, U‐73122 (phospholipase C (PLC) inhibitor), Gö6976 (protein kinase C (PKC) inhibitor), BAPTA‐AM (calcium chelator), LFM‐A13 (Bruton's tyrosine kinase (Btk)‐specific inhibitor), and PP2 (c‐Src kinase inhibitor) suppressed AmB‐induced phosphorylation of p65 and translocation of p65 into the nucleus. U‐73122 and Gö6976 reduced AmB‐mediated induction of proinflammatory cytokines (tumor necrosis factor (TNF)‐α and interleukin (IL)‐6) in RAW264.7 cells. Furthermore, AmB‐induced activation of NF‐ κ B was observed in toll‐like receptor (TLR) 2‐expressed cells, and the activation of NF‐κB was inhibited by U‐73122, whereas peptidoglycan‐induced NF‐κB activation, which was also dependent on TLR2, was not inhibited by U‐73122. Finally, U‐73122 partially suppressed in vivo production of TNF‐α and IL‐6 induced by AmB administration in BALB/c mice. These results suggested that the signaling from AmB stimulation to proinflammatory cytokine production is mediated by TLR2, Btk, PLC, PKC, c‐Src and NF‐κB. These signaling molecules may become a target for chemotherapy suppressing AmB‐induced proinflammatory cytokine production.     

Modulation of TXA2 generation of platelets by human lipoproteins

The lipoprotein (LP) fractions VLDL, LDL, HDL2 and HDL3 were prepared by ultracentrifugation of plasma from healthy volunteers and from patients with coronary heart disease (CHD). We investigated the capacity of platelets from healthy volunteers and patients with atherosclerosis to generate thromboxane A2 (TXA2) during spontaneous clotting of whole blood under the influence of the lipoprotein fractions. In our experiments the serum concentration of TXB2, reflecting the capacity of platelets to generate TXA2 during clotting, depends on several factors: the type of LP fraction used, the blood used for generation of TXA2, and for the same LP fraction whether it was taken from plasma of healthy volunteers or patients with CHD. VLDL prepared from plasma of healthy volunteers inhibited but VLDL prepared from plasma of patients with CHD enhanced the TXA2 formation of platelets from healthy volunteers (p less than 0.05, resp.). LDL from CHD patients inhibited the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01). The HDL subfractions HDL2 and HDL3 from healthy volunteers inhibited TXA2 formation by platelets from healthy volunteers as well as those from atherosclerotic patients (p less than 0.05; p less than 0.01, respectively). HDL2 from patients with CHD inhibited only the TXA2 formation of platelets from healthy volunteers (p less than 0.01), whereas HDL3 from CHD patients inhibited only the TXA2 formation of platelets from atherosclerotic patients (p less than 0.01).     

Inhibition of the interaction between lipoproteins and amphotericin B by some delivery systems.

Amphotericin B (AmB) is a potent antifungal agent used to treat patients with systemic mycoses. The clinical usefulness of the drug is limited by its high toxicity and several new less toxic formulations of AmB have been recently developed. In order to understand the mechanism of the decreases of toxicity caused by various new delivery systems, we have investigated by uv-visible spectroscopy the interaction of two of these formulations with human blood lipoproteins. The results were compared with those obtained with the commonly used pharmaceutical form of AmB (Fungizone). This study shows that AmB-lipoprotein interaction is hindered when the drug is in a monomeric form and/or when it is included in phospholipid-surfactant micelles. In an in vivo study on mice it is shown here that AmB monomerized by surfactant is less toxic to animals than the same concentration of Fungizone, where the polyene is strongly aggregated. It may be concluded from the present study that the AmB species which is responsible for the in vivo toxicity is a complex of the antibiotic with the low density and the very low density blood lipoproteins and that hindering of this complex formation results in a decrease of AmB toxicity.     

Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins

Peroxynitrite has been implicated in the oxidative modification of low-density lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO(2)-lipoprotein) and investigated the effect of NO(2)-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AII, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO(2)-HDL, as quantitated by HPLC, and the amount in NO(2)-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO(2)-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NO(2)-lipoproteins. Incubation with either NO(2)-HDL or NO(2)-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO(2)-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO(2)-LDL significantly reduced the expression and activity of Cu(2+),Zn(2+)-superoxide dismutase (CuZn-SOD) in the cells. Neither NO(2)-HDL nor NO(2)-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production in NO(2)-lipoprotein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO(2)-lipoprotein. Overall, NO(2)-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses.     

In vivo immunostimulation by tuftsin

Summary Tuftsin, a physiological basic tetrapeptide known for its capacity to stimulate the phagocytic activity of macrophages and polymorphonuclear leucocytes, was examined for its immunostimulating properties when injected systemically into mice.Tuftsin was able to potentiate the antibody response to a thymus-dependent antigen (TNP-KLH) when injected 3, 7 or 10 days before the antigen. The response to the same hapten coupled to a thymus-independent carrier (LPS) was stimulated on day 1 and 3 but slightly depressed on day 7.The peptide rendered macrophages highly cytostatic for tumor cells but the activation process required 7 days to develop. In contrast, enhancement of antibody-dependent cell-mediated cytotoxic (ADCC) activity of spleen cells was observed throughout the period of observation (14 days).Tuftsin did not induce nonspecific suppressor cells as the response of normal spleen cells to mitogens in vitro was not depressed when cultivated in the presence of tuftsin-treated spleen cells.These observations suggest that tuftsin acts as an immunostimulant which may be useful in cancer immunotherapy.This work was supported by grants from DGRST (n^o 78.7.2651) and from INSERM (contract libre n^o 78.5.168.2).     

A kinetic study of the oxidation effects of amphotericin B on human low-density lipoproteins

The UV-visible results of this kinetic study show that amphothericin B as Fungizone is a much stronger oxidant than CuSO(4), itself a powerful oxidant of low-density lipoprotein (LDL). Amphotericin B as AmBisome alone has no oxidizing effect on LDL while a mixture of both AmBisome and CuSO(4) induces an important potentialization of the LDL oxidation. These results allow us to believe that the high toxicity of amphotericin B is related to its capacity to modify and to weaken the structure of LDL. In addition, differential scanning calorimetry experiments show that the oxidative modifications of LDL by CuSO(4) or by amphotericin B proceed through different mechanisms.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.     

Resistance of the neck plasma membrane between the mother and the bud of Saccharomyces cerevisiae and Candida albicans to amphotericin B-induced deformation

Abstract The polyene antibiotic, amphotericin B, at high concentrations (5–20 μg/ml) induced particle-free smooth areas in the plasma membranes of Saccharomyces cerevisiae and Candida albicans . These areas occured more or less over the entire plasma membrane of unbudded cells. In budded cells, however, the neck between the mother and bud did not undergo deformation. This suggests the strong interaction between the filamentous ring, which is firmly attached to the neck plasma membrane, and plasma membrane particles in the neck regions.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes

Intact erythrocytes incubated in the presence of low density lipoproteins (LDL) undergo a time-dependent morphologic transformation from biconcave discs to spherocytes within 4 h. No shape change is observed when erythrocytes are incubated with high density lipoproteins (HDL). The LDL-induced change in erythrocyte morphology occurs without concomitant leakage of hemoglobin from the cell or depletion of intracellular ATP; no change in the distribution of the major lipids of the erythrocyte membranes was detected. The alteration of morphology does require attachment of LDL to the erythrocyte surface. The LDL-induced morphologic alteration is inhibited by HDL, but not by serum albumin. HDL prevent the attachment of LDL to the cell membrane; however, the HDL subfractions, HDL₂ and HDL₃, are only partially effective. These data suggest that normal erythrocyte morphology and cell function may depend on the concentration and composition of the circulating lipoproteins.     

Circular dichroism study of interactions of Fungizone or AmBisome forms of amphotericin B with human low density lipoproteins

Amphotericin B (AmB), a potent antifungal agent used to treat invasive fungal infections, is still employed more than 40 years after its introduction in the pharmacopea. When injected into the blood stream, this antibiotic is carried by low density lipoproteins (LDLs) to which it induces the formation of oxidation products responsible in part for some of the severe adverse effects of the drug. However, the oxidative damages induced to LDLs are not yet understood. We present here the effects of the Fungizone and AmBisome forms of AmB on LDLs as compared to those of CuSO(4), a well-known powerful oxidant of LDLs. We use circular dichroism (CD) spectroscopy, which is particularly useful because it allows the investigation of the structural integrity of the proteic moiety of LDL upon interaction with AmB. The CD spectra also yield information on the drug itself because in its oligomer form it presents a strong dichroic signal in a spectral region different from that of the protein. Our results show that neither form of AmB changes the secondary structure of the protein while the helical content of the LDL is increased either in the presence of CuSO(4) alone or in the presence of CuSO(4) and AmBisome or Fungizone. On the other hand, the CD spectra of the antibiotic indicate that Fungizone AmB suffers important oxidative damage in the presence of LDLs and CuSO(4) while this damage is not present with AmBisome AmB. These observations lead us to propose that the structural modifications of the proteic part of LDLs induced by the Cu(2+) ions are involved in the important oxidative damage suffered by Fungizone AmB, which in this form is much more susceptible to interaction with its environment than AmBisome.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

Interaction of plasma lipoproteins with erythrocytes. II. Modulation of membrane-associated enzymes.

When incubated with intact erythrocytes, low density lipoproteins (LDL) decrease the phosphate content of erythrocyte spectrin allowing the cells to undergo morphological transformation. The phosphate content of spectrin depends on the balance between the activity of membrane-associated cyclic AMP-independent protein kinases and phosphoprotein phosphates. LDL do not influence the activity of membrane-associated cyclic AMP-independent protein kinases; these lipoproteins activate by 2-fold and greater membrane-associated phosphatases as determined by hydrolysis of p-nitrophenyl phosphate and by phosphate hydrolysis of phosphorylated erythrocyte membrane proteins. We conclude that LDL interact at the exterior surface of the erythrocyte to stimulate dephosphorylation of spectrin. The significance of this conclusion is augmented by the fact that spectrin, the target for LDL-induced dephosphorylation, specifies cell morphology and modulates the distribution of cell-surface receptors. LDL also render erythrocyte acetylcholinesterase less susceptible to inhition by F-. Lipoproteins in the high density class (HDL) do not stimulate dephosphorylation of spectrin, and they are consequently unable to alter erythrocyte morphology. HDL do prevent the LDL-induced activation of membrane phosphatase. The inhibitory capacity of HDL is observed over the range of LDL:HDL (w/w) which exists in the plasma of normolipemic humans.     

Alteration of membrane permeability by amphotericin B

Amphotericin B (AmB) increased unidirectional Na transport and net transcellular sodium movements across the skin of the frog, Rana pipiens, when added to the solution bathing the corium side, but not from the outer epidermal surface. The AmB response was prevented with pretreatment with amiloride, ouabain and mucosal sodium substitution. Alteration in pH markedly reduced the permeability changes induced by AmB. AmB did not interfere with the increase in sodium transport induced by antidiuretic hormone. The present study demonstrates that AmB interacts with the skin of the frog, Rana pipiens, from the corium side specifically increasing transepithelial sodium transport. The increase in transport apparently occurs through the existing sodium pathway.     

Studies on immunity to Babesia gibsoni in dogs immunostimulation by Bordetella bronchiseptica

Four species of bacteria, Corynebacterium anaerobium 578, Actinobacillus pleuropneumoniae G-4, Mycobacterium bovis BCG, and Bordetella bronchiseptica A-2, were injected intravenously into mice (5 weeks old, ICR-SPF). The clearance of carbon from the blood stream and the weights of the spleen and liver were determined as indicators of RES stimulation. Mouse footpad reaction was assessed as an indicator of delayed-type hypersensitivity to each species of bacteria. The immuno-stimulative activity of each species of bacteria against bovine serum albumin was monitored by passive hemagglutination assay and the macrophage migration-inhibition test in guinea pigs. Based on the results of the experiments described above, B. bronchiseptica was selected as an immunostimulator (Ims) for immunization trials of the hemo-protozoan parasite, Babesia gibsoni, with inactivated merozoites of B. gibsoni (BgK). Twelve dogs, pointers about 6 months old, were divided into four groups of three dogs each. Group 1 dogs were initially injected with Ims, and later injected with BgK and Ims (BgK+Ims) after a 3-week interval. Group 2 and Group 3 dogs were injected twice, at a 3-week interval, with BgK+Ims and BgK, respectively, and Group 4 served as a control. As the results, the serum antibody titres of Group 1 and 2 were several times higher than that of Group 3, and the cell-mediated immunity to parasites was noticeably stimulated by immunization with BgK+Ims. The peak level of parasitemia following the challenge were over 10% for Group 4 and 4.5% for Group 3, while levels for Group 1 and 2 were 2.5% and less than 1%, respectively. No such major clinical signs of babesiosis as jaundice and anemia were observed in Group 1 or 2.     

Mobilization of heparan sulfate induced by immunostimulation in a patient with mucopolysaccharidosis IIIA

Unspecific immunostimulation by bacterial vaccines of a patient with mucopolysaccharidosis IIIA (Sanfilippo A) syndrome induces a marked increase in the urinary excretion of heparan sulfate and of uronatecontaining oligosaccharides. This event is presumably linked to an increased vascular permeability and exocytosis of storage material, elicited by mediators of inflammation, as well as to enhanced degradation of stored polymers in activated macrophages and surrounding tissue.     

Inhibition of photosynthetic electron transport by amphotericin B

By assaying partial reactions of the photosynthetic electron transport system using thylakoids from spinach as well as from the algae Bumilleriopsis, Dunaliella , and Anabaena , it was demonstrated that the polyene antibiotic amphotericin B has no specific effect on plastocyanin. Pretreating spinach and algal thylakoids with this antibiotic decreased photosystem-II as well as photosystem-I activity regardless of whether the membranes contained plastocyanin or cytochrome c-553. Different sensitivity of cell-free electron transport activity against this antibiotic was observed due to the species used. With Dunaliella , the photosystem-II region was inhibited more strongly than photosystem-I, while Bumilleriopsis chloroplasts – although not containing plastocyanin – exhibited a stronger inhibition of the photosystem-I region. Apparently, amphotericin B mainly solubilizes redox compounds that form connecting pools in the photosynthetic electron transport chain.