Inhibition of Nucleotide Biosynthesis Potentiates the Antifungal Activity of Amphotericin B

The polyene antifungal agent Amphotericin B exhibits potent and broad spectrum fungicidal activity. However, high nephrotoxicity can hinder its administration in resource poor settings. Quantification of early fungicidal activity in studies of HIV patients with cryptococcosis demonstrate that 5-Fluorocytosine therapy in combination with Amphotericin B results in faster clearance than with Amphotericin B alone. In vitro synergy between the two drugs has also been reported but the mechanism by which 5-Fluorocytosine synergizes with Amphotericin B has not been delineated. In this study we set out to investigate the effect of genetic mutation or pharmacologic repression of de novo pyrimidine and purine biosynthesis pathways on the Amphotericin B susceptibility of Cryptococcus neoformans. We demonstrate that a ura- derivative of wild type Cryptococcus neoformans strain H99 is hypersensitive to Amphotericin B. This sensitivity is remediated by re-introduction of a wild type URA5 gene, but not by addition of exogenous uracil to supplement the auxotrophy. Repression of guanine biosynthesis by treatment with the inosine monophosphate dehydrogenase inhibitor, mycophenolic acid, was synergistic with Amphotericin B as determined by checkerboard analysis. As in Cryptococcus neoformans, a ura ⁻ derivative of Candida albicans was also hypersensitive to Amphotericin B, and treatment of Candida albicans with mycophenolic acid was likewise synergistic with Amphotericin B. In contrast, neither mycophenolic acid nor 5-FC had an effect on the Amphotericin B susceptibility of Aspergillus fumigatus. These studies suggest that pharmacological targeting of nucleotide biosynthesis pathways has potential to lower the effective dose of Amphotericin B for both C. neoformans and C. albicans. Given the requirement of nucleotide and nucleotide sugars for growth and pathogenesis of Cryptococcus neoformans, disrupting nucleotide metabolic pathways might thus be an effective mechanism for the development of novel antifungal drugs.     

Freeze-etch electron microscopy of erythrocytes,Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B

Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150–250Åin diameter, in the membranes of rat erythrocytes, in cholesterol-containing membranes ofAcholeplasma laidlawii cells and in egg lecithin-cholesterol liposomes. No change in fracture faces was observed when cholesterol was absent in the membranes ofA. laidlawii, and lecithin liposomes.Amphotericin B does not visibly affect the freeze-etch morphology of erythrocytes, cholesterol-containingA. laidlawii cells and lecithin-cholesterol liposomes.     

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.     

Mucor irregularis Infection Around the Inner Canthus Cured by Amphotericin B: A Case Report and Review of Published Literatures

We report a case of primary cutaneous mucormycosis caused by Mucor irregularis. A 47-year-old farmer was presented to our clinic with the history of progressive red plaque around the inner canthus following dacryocystectomy about a year earlier. Linear, aseptate hyphae were seen by direct KOH examination and in biopsy. Fungal culture revealed light yellow filamentous colonies that were identified as Mucor irregularis by nucleotide sequencing of rRNA gene. Amphotericin B and dexamethasone were used in gradually increasing dosage. The treatment lasted 43 days, and the patient received 760 mg total amphotericin B. The patient was discharged after 2 months of treatment. The plaque became smooth, and fungal culture was negative. There was no recurrence for half a year through telephone follow-ups. A review of published studies revealed 23 cases of Mucor irregularis infection. Most cases resulted following injuries or surgical complications. Farmers and manual laborers were most at risk with males outnumbering females among patients. Amphotericin B and its liposomal preparations remain most effective treatment choices.     

Determination of free and liposomal Amphotericin B in human plasma by liquid chromatography–mass spectroscopy with solid phase extraction and protein precipitation techniques

Amphotericin B is available in various drug delivery systems such as cholesteryl sulfate complex, as lipid complex, and as liposomal formulation. The separation and measurement of free drug (drug which is not bound with liposomal lipids) and liposomal drug (drug which is entrapped in liposomes) in the human plasma after injection of liposomal Amphotericin B is of prime importance due to toxicity concerns. A robust, specific and sensitive method has been developed to effectively separate and then quantify the free drug and liposomal drug, present in human plasma. This method utilizes solid phase extraction Oasis HLB cartridges, which retains the free drug and the liposomal Amphotericin B was eluted from the cartridge in first step. The eluted liposomal Amphotericin B was then extracted from lipids by protein precipitation method using 2% dimethylsulfoxide (DMSO) in acetonitrile. After separation and extraction, the quantification of free and liposomal fractions of Amphotericin B was performed by HPLC–MS–MS technique. The chromatographic separation was performed using Chromolith Performance RP 18e column. The mobile phase composed of 5 mM ammonium acetate, methanol and acetonitrile and a gradient elution program was used. The calibration curves were found to be linear for free Amphotericin B (0.25–15.0 μg/ml) and liposomal Amphotericin B (1.0–100.0 μg/ml). The recovery was about 96% for free Amphotericin B and about 92% for liposomal Amphotericin B. Recoveries were consistent over the linearity ranges defined. The intra-batch and inter-batch accuracy and precision fulfilled the international requirements. The stability of free and liposomal Amphotericin B was assessed under different storage conditions.     

The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.     

The importance of different murine cell types in the immunopotentiation produced by amphotericin methyl ester.

Amphotericin R (AmB) or its methyl ester (AME) are potent stimulants of humoral and cell-mediated immunity in mice. When AME is injected simultaneously with antigen, it augments humoral immunity by expansion of the primary as well as the memory B cell pool. The most consistent and dramatic effect of amphotericin on humoral immunity is the enhancement of secondary IgG responses. This effect on the IgM-IgG switch mechanism depends on T cells and is reduced following thymectomy on adult mice. The route and timing of amphotericin administration also have dramatic quantitative, effects on immunopotentiation. Neither polyclonal B cell activation nor stimulation of antigen uptake in vitro by peritoneal macrophages was observed following intraperitoneal injection of AME. A tentative hypothesis is that antigen-stimulated T cells are important sites of the immunostimulant effects of AME on murine immune responses.     

Electrochemical Measurement of Electron Transfer Kinetics by Shewanella oneidensis MR-1

Shewanella oneidensis strain MR-1 can respire using carbon electrodes and metal oxyhydroxides as electron acceptors, requiring mechanisms for transferring electrons from the cell interior to surfaces located beyond the cell. Although purified outer membrane cytochromes will reduce both electrodes and metals, S. oneidensis also secretes flavins, which accelerate electron transfer to metals and electrodes. We developed techniques for detecting direct electron transfer by intact cells, using turnover and single turnover voltammetry. Metabolically active cells attached to graphite electrodes produced thin (submonolayer) films that demonstrated both catalytic and reversible electron transfer in the presence and absence of flavins. In the absence of soluble flavins, electron transfer occurred in a broad potential window centered at ∼0 V (versus standard hydrogen electrode), and was altered in single (ΔomcA, ΔmtrC) and double deletion (ΔomcA/ΔmtrC) mutants of outer membrane cytochromes. The addition of soluble flavins at physiological concentrations significantly accelerated electron transfer and allowed catalytic electron transfer to occur at lower applied potentials (−0.2 V). Scan rate analysis indicated that rate constants for direct electron transfer were slower than those reported for pure cytochromes (∼1 s⁻¹). These observations indicated that anodic current in the higher (>0 V) window is due to activation of a direct transfer mechanism, whereas electron transfer at lower potentials is enabled by flavins. The electrochemical dissection of these activities in living cells into two systems with characteristic midpoint potentials and kinetic behaviors explains prior observations and demonstrates the complementary nature of S. oneidensis electron transfer strategies.Respiratory electron flow typically occurs at the inner membrane, where oxidation and reduction can be easily linked to intracellular electron carriers and used to generate a membrane potential. However, when the electron acceptor or donor is insoluble, bacteria must possess a mechanism for transferring electrons beyond their inner membrane (1). This is especially true for Proteobcteria, which have an outer membrane that further insulates cytoplasmic and inner membrane processes from insoluble substrates. Metal oxides (such as Fe(III) and Mn(IV) oxyhydroxides) are well recognized naturally occurring electron acceptors that demand such an electron transfer strategy (2–4).Shewanella oneidensis MR-1, a metabolically versatile member of the gammaproteobacteria (5), is capable of reducing insoluble metals, and this phenotype has been linked to a collection of interacting multiheme cytochromes spanning the inner membrane, periplasmic space, and outer membrane (6–12). There is also evidence that some of these cytochromes decorate the surface of pili-like structures extending from the cell surface (13, 14). Regardless of the ultimate location of the cytochromes, in all models of electron transfer, electrons must hop from these proteins to a solid surface or be transferred to a soluble mediator that can diffuse to a final destination (15, 16). Although chelation of a metal oxide is a third option (17, 18), the fact that Shewanella is able to transfer electrons to solid graphite electrodes (19–23) underscores the need for a direct or diffusion-based electron transfer mechanism to link cellular proteins and surfaces.Recent work has shown that Shewanella species secrete soluble flavins (FMN and riboflavin) that facilitate electron transfer to both metals and electrodes (23, 24). For example, removal of accumulated soluble flavins decreases the rate of electron transfer by Shewanella biofilms to electrodes over 80%. Consistent with this observation, kinetic measurements with pure MtrC and OmcA (25) showed that direct reduction of solid metal oxides by these cytochromes was too slow to explain physiological rates of electron transfer, whereas turnover rates of these enzymes with soluble flavins were orders of magnitude larger. These studies suggest that the kinetics of electron transfer from cytochromes on the outer surface of Shewanella to electrodes will be significantly altered in the absence of diffusible mediators (9–11, 26–34).Voltammetry has proven a useful technique for the analysis of electron transfer rates and pathways using purified proteins (35–39) and has recently been extended to the study of intact bacteria (23, 40–42). In slow scan rate cyclic voltammetry, the rate of electron transfer from respiring Shewanella biofilms to electrodes rises sharply at the E°′ of riboflavin and FMN (−0.2 V versus SHE)² (23). Such measurements relating thermodynamic driving force to turnover kinetics would be difficult with whole cell:Fe(III) oxide incubations, which do not allow fine control over the electron acceptor redox potential or real time recording of electron transfer rates. In addition, voltammetry provides tools to observe electron movement under single-turnover conditions (in the absence of electron donor), allowing reversible oxidation and reduction of proteins accessible to the electrode and study of kinetic behavior (43, 44).In this work, techniques of turnover (sustained electron transfer from cells to electrode in the presence of electron donor) and single turnover (reversible oxidation and reduction in the absence of electron donor) voltammetry were harnessed to investigate the role of outer membrane proteins in electron transfer from Shewanella to electrodes. In all of these studies, intact metabolically active cells were used, along with electrode surfaces known to act as acceptors for Shewanella. The results in the absence of soluble mediators provide evidence that electron transfer between MtrC and OmcA and surfaces requires a higher potential compared with when flavins are present to shuttle electrons to the surface. Mutant analysis also demonstrates that cells possessing different outer membrane cytochromes have differing abilities for direct and mediator-enabled electron transfer.     

Synthesis and in vitro biological evaluation of novel coumarin derivatives containing isoxazole moieties on melanin synthesis in B16 cells and inhibition on bacteria

A novel series of coumarin derivatives 6a–o, bearing isoxazole moieties were designed and synthesized. After that, they were evaluated for melanin synthesis in murine B16 cells and inhibitory effect on the growth of CA (Candida albicans), EC (Escherichia coli), SA (Staphylococcus aureus). It was found that eleven compounds (6b–f, 6j–o) showed a better activity on melanin synthesis than positive control (8-MOP). Among them, compounds 6d (242%) and 6f (390%), with nearly 1.6 and 2.6-fold potency compared with 8-MOP (149%) respectively, were recognized as the most promising candidate hits for further pharmacological study of anti-vitiligo.Seven halogen substituted compounds exhibited moderate antimicrobial activity against CA. It is interesting that 6e–f and 6l–m, which had two halogens on the benzene showed a comparable activity with Amphotericin B against CA.The evaluation of melanin synthesis in B16 cells and inhibitory effect on bacteria of above structurally diverse derivatives had also led to an outline of structure-activity relationship.     

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-γ in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.     

Differential Damage in Bacterial Cells by Microwave Radiation on the Basis of Cell Wall Structure

Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.     

Electrolyte transport by gallbladders of rabbit and guinea pig: Effect of amphotericin B and evidence of rheogenic Na transport

Summary Ion transport and electrical properties of rabbit and guinea pig gallbladders were investigated to gain further information about the active transport mechanism that mediates fluid absorption. The intracellular and transepithelial electrical potentials were measured simultaneously using the microelectrode technique. Exposure of the mucosal surface to Amphotericin B resulted in the prompt development of a serosa-positive electrical potential difference (PD) which could not be attributed to an alteration in ion diffusion potentials across either the cell membrane or across the tight junction. Because the Amphotericin B-inducedPD was immediately dependent on warm temperatures and O₂, and was independent of NA and K concentration gradients across the cell membrane, it is suggested that active ion transport is directly responsible for thePD. Since thePD was abolished in the absence of Na in the bathing solutions, a rheogenic Na pump is postulated; this pump also appears to be operative in tissue not exposed to Amphotericin B. The specific tissue properties altered by Amphotericin B to produce a serosa-positivePD remain incompletely defined. The results of the present study indicate that ion transport by rabbit gallbladderin vitro is a consequence of a rheogenic active Na transport mechanism at the basolateral membranes which, in conjunction with a coupled NaCl influx process at the mucosal border, ultimately results in absorption of NaCl and water.     

Central venous catheter related infections: Risk factors and the effect of glycopeptide antibiotics

Background

Invasive Aspergillus infections are frequently seen in immunocompromised patients but arthritis is a rare complication of Aspergillus infections in the absence of immune suppressive therapy, trauma or surgical intervention.

Case presentation

A 17 years old male patient with arthritis and patellar osteomyelitis of the left knee whose further investigations revealed chronic granulomatous disease as the underlying disease is followed. Aspergillus fumigatus was isolated from the synovial fluid and the tissue samples cultures. He was treated with Amphotericin B deoxicolate 0.7 mg/kg/day. Also surgical debridement was performed our patient. Amphotericin B nephrotoxicity developed and the therapy switched to itraconazole 400 mg/day. Itraconazole therapy were discontinued at the 6th month. He can perform all the activities of daily living including.

Conclusion

We think that, chronic granulomatous disease should be investigated in patients who have aspergillar arthritis and osteomyelitis.     

The inhibition of Candida species by selected essential oils and their synergism with amphotericin B

In this work we highlight a possible synergistic anti-Candida effect between Melaleuca alternifolia, Origanum vulgare and Pelargonium graveolens essential oils and the antifungal compound Amphotericin B. The antifungal activity was assessed using the agar dilution method in eleven Candida strains. The results obtained indicate the occurrence of a synergistic interaction between the essential oils under study and Amphotericin B. P. graveolens essential oil appeared to be the most effective, inhibiting all the Candida species evaluated by this study.     

Fluorescence probes in metastatic B16 melanoma membranes

Fluorescence probe molecules, trans-parinaric acid and 1,6-diphenylhexatriene, were utilized to characterize the structure of plasma membranes, microsomes and mitochondria from B16 melanoma cells. High metastatic B16-F10 and low metastatic B16-F1 melanoma cell lines had markedly different membrane structures. The fluorescence polarization, fluorescence lifetime and limiting anisotropy of trans-parinaric acid were significantly lower ($\text{P < 0.05}$) in all three membrane fractions of the B16-F1 cell line than in the corresponding membranes of the B16-F10 cell line. These data indicated less restriction to rotational motion in the solid lipid domains of B16-F1 cell membranes preferentially sensed by trans-parinaric acid. The limiting anisotropy of both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene was significantly lower in the outer monolayer than the inner monolayer of the plasma membrane of B16-F1 cells but not in B16-F10 cells. A breakpoint in Arrhenius plots of fluorescence near 30–34°C indicated the presence of a phase separation that was assigned to the inner monolayer of the plasma membrane. However, no differences in this breakpoint temperature were noted between the B16-F1 and B16-F10 melanoma membranes. Thus, more fluid solid membrane domains and a distinct transbilayer fluidity difference were characteristic of plasma membranes from low metastatic B16-F1 melanoma cells in contrast to high metastatic B16-F10 melanoma cells.     

Effect of amphotericin B on the infection success of Schistosoma mansoni in Biomphalaria glabrata

In the present study, we examined the effect of amphotericin B on larval stages (miracidia and primary sporocyst) of the helminth Schistosoma mansoni, the causative agent of human schistosomiasis. Amphotericin B (AmB) is a polyene macrolide that disturbs the function of the cell membrane; it is widely used as prophylactic antimycotic agent in in vitro culture. We show for the first time that S. mansoni miracidia infectivity is considerably reduced after AmB treatment. Moreover we demonstrate that AmB does not affect the development, growth, viability, and behavior of miracidia and primary sporocysts. Our data indicate that AmB effects on S. mansoni sporocyst prevalence are linked to the oxidative properties of AmB. These may alter the capacity of sporocysts to respond to the oxidative stress generated by the snail immune defence system.     

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell and murine macrophage susceptibility to axenic amastigotes for the screening of leishmanicidal compounds

This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 parasites/murine macrophage. The parasite burden was maximal at 72 h post-infection (h.p.i.) for THP-1 cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i. Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 μM in THP-1 and murine macrophages at 72 h.p.i.Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macrophages and indicate the suitability of this model to screen compounds for leishmanicidal activity.     

UBE2L3 Polymorphism Amplifies NF-κB Activation and Promotes Plasma Cell Development,Linking Linear Ubiquitination to Multiple Autoimmune Diseases

UBE2L3 is associated with increased susceptibility to numerous autoimmune diseases, but the underlying mechanism is unexplained. By using data from a genome-wide association study of systemic lupus erythematosus (SLE), we observed a single risk haplotype spanning UBE2L3, consistently aligned across multiple autoimmune diseases, associated with increased UBE2L3 expression in B cells and monocytes. rs140490 in the UBE2L3 promoter region showed the strongest association. UBE2L3 is an E2 ubiquitin-conjugating enzyme, specially adapted to function with HECT and RING-in-between-RING (RBR) E3 ligases, including HOIL-1 and HOIP, components of the linear ubiquitin chain assembly complex (LUBAC). Our data demonstrate that UBE2L3 is the preferred E2 conjugating enzyme for LUBAC in vivo, and UBE2L3 is essential for LUBAC-mediated activation of NF-κB. By accurately quantifying NF-κB translocation in primary human cells from healthy individuals stratified by rs140490 genotype, we observed that the autoimmune disease risk UBE2L3 genotype was correlated with basal NF-κB activation in unstimulated B cells and monocytes and regulated the sensitivity of NF-κB to CD40 stimulation in B cells and TNF stimulation in monocytes. The UBE2L3 risk allele correlated with increased circulating plasmablast and plasma cell numbers in SLE individuals, consistent with substantially elevated UBE2L3 protein levels in plasmablasts and plasma cells. These results identify key immunological consequences of the UBE2L3 autoimmune risk haplotype and highlight an important role for UBE2L3 in plasmablast and plasma cell development.