Continuous dosing of nikkomycin Z against systemic candidiasis, in vivo and in vitro correlates.

BACKGROUND: Candidiasis is the most common cause of fungal sepsis, and new agents are of interest to ameliorate current deficiencies in therapy. Nikkomycin Z (NIKZ) is an inhibitor of chitin synthase, interfering with fungal cell wall development. OBJECTIVES/METHODS: We studied NIKZ therapy of disseminated murine candidiasis, via continuous drug exposure, in drinking water, to compensate for rapid clearance of the drug. RESULTS: Drinking, and thus drug intake in the NIKZ groups, as well as body weight, was affected by the degree of illness. NIKZ effect on survival, despite reduced drinking initially after infection, was highly efficacious and dose-related, and comparable to fluconazole, though neither were curative with the regimens employed. The challenge was rapidly lethal to all untreated animals, whereas NIKZ groups achieved >50% survival. Assays of residual fungal infection were consistent with impressions of efficacy based on survival. Although NIKZ MIC for Candida albicans appeared unpromising, mycelial formation assays more closely correlated with in vivo observations. CONCLUSIONS: In vitro-in vivo disparity may be explained by NIKZ tissue concentration in the target tissue and/or by enhanced NIKZ action on mycelial formation, a morphological change in vivo wherein chitin synthesis is more critical, compared to NIKZ activity in inhibiting planktonic growth. A sustained release oral form of NIKZ in drug development for humans could hold promise, possibly also in future exploring previously demonstrated synergy in vitro with other antifungals.

Synergy In Vitro of Nikkomycin Z with Azole Against the Invasive Form of Candida albicans.

In a previous study, therapeutic activity of nikkomycin Z (NZ) in a model of invasive candidiasis did not appear to correlate with lesser activity in vitro (using classical MIC methods) with planktonic organisms. However, NZ potency was much greater assaying activity in vitro against germ tubes, the initiator of the invasive mycelial form of the fungus, as occurs in infected tissues. Synergy has been demonstrated for NZ and other drugs, notably fluconazole (the most commonly used drug against candidiasis), in planktonic testing, which correlated with results in vivo. This raised the question whether activity shown by NZ alone against germ tubes would be reflected in drug combinations, and even whether synergy testing against germ tubes might be a better correlate of synergy in future in vivo studies. We show in this study significant NZ synergy with fluconazole against germ tubes, for several C. albicans isolates, with testing in many drug ratios. This observation opens the way for further explorations of this method of susceptibility testing for synergy, and correlation with combination therapy against candidiasis.

Nikkomycin Z against Disseminated Coccidioidomycosis in a Murine Model of Sustained-Release Dosing.

Nikkomycin Z (nikZ) is a chitin synthase inhibitor. Efficacy against Coccidioides has been demonstrated in animal models of pulmonary or brain infection. Its short half-life in mice and in humans would necessitate divided daily dosing. We assayed nikZ efficacy in disseminated coccidioidomycosis (in a reduction of CFU design) and whether sustained release might be useful. Mice were challenged intravenously with low or high arthroconidial inocula. Fluconazole, clinically the most commonly used anticoccidioidal drug, was compared (gavage) at high dose to a dose range of nikZ administered intraperitoneally or, to mimic sustained release, administered continuously in drinking water. Therapy was given for 5 days. In vitro, both fluconazole and nikZ inhibited the isolate studied; nikZ was fungicidal. Oral nikZ therapy gave similar results to intraperitoneal nikZ and sterilized infection in most animals after low-inoculum challenge. In both challenges, oral nikZ produced greater reduction of CFU in organs (lung, liver, and spleen) than fluconazole. Oral nikZ doses of ≥200 mg/kg of body weight/day were particularly effective in all organs and were well tolerated. This efficacy occurred even though, after severe challenge, mice had reduced water intake, resulting in ingesting less than the desired dose, particularly initially after infection. This study shows, for the first time, efficacy of nikZ against disseminated coccidioidomycosis. Efficacy was shown after challenges producing different levels of severity of disease. This study also suggests the likely benefits of developing an extended release formulation supplying continuous systemic concentrations of nikZ.

Efficacy and Associated Drug Exposures of Isavuconazole and Fluconazole in an Experimental Model of Coccidioidomycosis.

Coccidioides spp. are important pathogens in regions where they are endemic, and new treatment options are needed. Here, isavuconazonium sulfate (ISAVUSULF) and fluconazole (FLU) were evaluated in experimental disseminated coccidioidomycosis to characterize drug exposures associated with efficacy. Broth macrodilution was performed on Coccidioides isolates to measure minimal effective concentrations (MEC) and minimal fungicidal concentrations (MFC). Mice were inoculated with Coccidioides posadasii (Silveira strain). Treatment started 4 days postinoculation. In model 1, mice were treated for 19 days, followed by 30 days of off-therapy observation, measuring survival through day 49 and residual fungal burden. Treatments included ISAVUSULF (prodrug; 186, 279, or 372 mg/kg twice daily), FLU (20 or 100 mg/kg once daily), and no treatment. Model 2 included 7-day treatment with ISAVUSULF (prodrug; 74.4, 111.6, or 148.8 mg/kg twice daily), FLU (20 or 100 mg/kg once daily), and no treatment. Serial plasma and tissues samples were obtained for pharmacokinetics (PK) and fungal burden measurement, respectively. Fifty percent minimal effective concentration (MEC50) values were 0.39 mg/liter (isavuconazole [ISAV]) and 12.5 mg/liter (FLU). Treatment with ISAVUSULF186 or with either FLU dose resulted in higher survival compared to that in the untreated group. Treatment with ISAVUSULF186 or ISAVUSULF279 twice daily or FLU100 reduced fungal burden in all organs (model 1). In model 2, a >1 log10 CFU/organ reduction was demonstrated, with ISAV area under the concentration-time curve (AUC) values achieved with 111.6 mg/kg twice daily (56.8 mg · h/liter) in the spleen and liver. FLU AUC values of 100 and 500 mg·h/liter for 20 and 100 mg/kg doses, respectively, resulted in a >1 log10 CFU/organ mean reduction in all organs. ISAVUSULF and FLU improved survival and reduced fungal burden. Increasing plasma drug exposures resulted in decreases in fungal burden.

Efficacy of nikkomycin Z in murine CNS coccidioidomycosis: modelling sustained-release dosing.

OBJECTIVES: Meningitis is the most feared coccidioidomycosis complication. Nikkomycin Z (nikZ) is a chitin synthase inhibitor. A concern is short half-life, necessitating multiple dose/day regimens. We simulated extended release, providing nikZ in drinking water. Extended release would enhance convenience, and adherence, for patients. METHODS: Coccidioides posadasii was injected intracerebrally into mice. Twelve day treatments began on Day 3. Fluconazole was given 100 mg/kg once daily (gavage); designed doses of nikZ 30, 100 or 300 mg/kg/day in drinking water. On Day 30 post-treatment, survivors were euthanized, brain cfu quantitated and cfu in other organs assessed. RESULTS: nikZ was stable in drinking water. Survival was 11%, 50%, 70%, 90% and 100% in untreated controls, fluconazole and nikZ 30, 100 and 300 mg/kg/day, respectively ; nikZ 300 mg/kg/day was superior (P ≤ 0.01) to fluconazole. Brains were sterilized in 0%, 20%, 86%, 89% and 80% of mice, respectively; nikZ 100 or 300 mg/kg/day was superior (P ≤ 0.01) to fluconazole. Clearance of infection in other organs was similar. All decreased drinking after infection, causing nikZ mice to ingest less than the desired dose in early therapy; despite this, they recovered sufficiently to resume pre-infection drinking and designed drug intakes. Thus, when sickest, even less than the designed dose was sufficient to enable recovery. CONCLUSIONS: This efficacy supports the development of sustained-release nikZ. Decreased intake wouldn't be a factor in humans, receiving drug via extended-release pill or continuous IV infusion. In prior studies (twice daily nikZ) of murine coccidioidal meningitis, results were inferior, suggesting sustained release may provide both convenience and superior outcomes.

Under nonlimiting iron conditions pyocyanin is a major antifungal molecule, and differences between prototypic Pseudomonas aeruginosa strains.

Airways of immunocompromised patients, or individuals with cystic fibrosis (CF), are common ground for Pseudomonas aeruginosa and Aspergillus fumigatus infections. Hence, in such a microenvironment both pathogens compete for resources. While under limiting iron conditions the siderophore pyoverdine is the most effective antifungal P. aeruginosa product, we now provide evidence that under nonlimiting iron conditions P. aeruginosa supernatants lack pyoverdine but still possess considerable antifungal activity. Spectrometric analyses of P. aeruginosa supernatants revealed the presence of phenazines, such as pyocyanin, only under nonlimiting iron conditions. Supernatants of quorum sensing mutants of strain PA14, defective in phenazine production, as well as supernatants of the P. aeruginosa strain PAO1, lacked pyocyanin, and were less inhibitory toward A. fumigatus biofilms under nonlimiting iron conditions. When blood as a natural source of iron was present during P. aeruginosa supernatant production, pyoverdine was absent, and phenazines, including pyocyanin, appeared, resulting in an antifungal effect on A. fumigatus biofilms. Pure pyocyanin reduced A. fumigatus biofilm metabolism. In summary, P. aeruginosa has mechanisms to compete with A. fumigatus under limiting and non-limiting iron conditions, and can switch from iron-denial-based to toxin-based antifungal activity. This has implications for the evolution of the microbiome in clinical settings where the two pathogens co-exist. Important differences in the iron response of P. aeruginosa laboratory strains PA14 and PAO1 were also uncovered.

P. aeruginosa (Pa) and A. fumigatus (Af) form biofilms in lungs of persons with cystic fibrosis and interact via virulence factors. Pa inhibits Af via different factors, depending on the availability of iron from blood. Low iron favors the use of pyoverdine, high iron the use of the toxin pyocyanin.

Novel intermicrobial molecular interaction: Pseudomonas aeruginosa Quinolone Signal (PQS) modulates Aspergillus fumigatus response to iron.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af), the commonest bacterium and fungus in compromised host airways, compete for iron (Fe). The Pseudomonas quinolone signal (PQS), a Pa quorum sensing molecule, also chelates Fe, and delivers Fe to the Pa cell membrane using Pa siderophores. In models of Af biofilm formation or preformed biofilms, PQS inhibited Af in a low Fe environment. AfΔsidA (mutant unable to produce siderophores) biofilm was more sensitive to PQS inhibition than wild-type (WT), as was planktonic AfΔsidA growth. PQS decreased WT Af growth on agar. All these inhibitory actions were reversed by Fe. The Pa siderophore pyoverdin, or Af siderophore inhibitor celastrol, act cooperatively with PQS in Af inhibition. These findings all indicate PQS inhibition is owing to Fe chelation. Remarkably, in high Fe environments, PQS enhanced Af biofilm at 1/100 to 1/2000 Fe concentration required for Fe alone to enhance. Planktonic Af growth, and on agar, Af conidiation, were also enhanced by PQS+Fe compared to Fe alone. In contrast, neither AfΔsidA biofilm, nor planktonic AfΔsidA, were enhanced by PQS-Fe compared to Fe. When Af siderophore ferricrocin (FC),+PQS, were added to AfΔsidA, Af was then boosted more than by FC alone. Moreover, FC+PQS+Fe boosted AfΔsidA more than Fe, FC, FC+Fe, PQS+FC or PQS+Fe. Thus PQS-Fe maximal stimulation requires Af siderophores. PQS inhibits Af via chelation under low Fe conditions. In a high Fe environment, PQS paradoxically stimulates Af efficiently, and this involves Af siderophores. PQS production by Pa could stimulate Af in cystic fibrosis airways, where Fe homeostasis is altered and Fe levels increase, supporting fungal growth.

Diphenyl diselenide and its interaction with antifungals against Aspergillus spp.

Given the few antifungal classes available to treat aspergillosis, this study aimed to evaluate the in vitro antifungal activity of diphenyl diselenide (PhSe)2 alone and in combination with classical antifungals against Aspergillus spp., and its in vivo activity in a systemic experimental aspergillosis model. We performed in vitro broth microdilution assay of (PhSe)2 against 32 Aspergillus isolates; and a checkboard assay to test the interaction of this compound with itraconazole (ITC), voriconazole (VRC), amphotericin B (AMB), and caspofungin (CAS), against nine Aspergillus isolates. An experimental model of invasive aspergillosis in mice was studied, and survival curves were compared between an untreated group and groups treated with 100 mg/kg ITC, or (PhSe)2 in different dosages (10 mg/kg, 50 mg/kg and 100 mg/kg). All Aspergillus non-fumigatus and 50% of A. fumigatus were inhibited by (PhSe)2 in concentrations ≤ 64 µg/ml, with significant differences in MICs between the sections. Synergism or additive effect in the in vitro (PhSe)2 interaction with VRC and CAS was observed against the majority of isolates, and with ITC against the non-fumigatus strains. In addition to the inhibitory interaction, (PhSe)2 was able to add a fungicidal effect to CAS. Survival curves from the systemic experimental aspergillosis model demonstrated that the inoculum caused an acute and lethal infection in mice, and no treatment applied significantly prolonged survival over that of the control group. The results highlight the promising activity of (PhSe)2 against Aspergillus species, but more in vivo studies are needed to determine its potential applicability in aspergillosis treatment. LAY SUMMARY: The activity of diphenyl diselenide (PhSe)2 alone and in combination with itraconazole, voriconazole, and caspofungin, is described against three of the most pathogenic Aspergillus sections. (PhSe)2 may prove useful in therapy of infection in future; further study is required.

Aspergillus-Pseudomonas interaction, relevant to competition in airways.

In airways of immunocompromised patients and individuals with cystic fibrosis, Pseudomonas aeruginosa and Aspergillus fumigatus are the most common opportunistic bacterial and fungal pathogens. Both pathogens form biofilms and cause acute and chronic illnesses. Previous studies revealed that P. aeruginosa is able to inhibit A. fumigatus biofilms in vitro. While numerous P. aeruginosa molecules have been shown to affect A. fumigatus, there never has been a systematic approach to define the principal causative agent. We studied 24 P. aeruginosa mutants, with deletions in genes important for virulence, iron acquisition, or quorum sensing, for their ability to interfere with A. fumigatus biofilms. Cells, planktonic or biofilm culture filtrates of four P. aeruginosa mutants, pvdD-pchE-, pvdD-, lasR-rhlR-, and lasR-, inhibited A. fumigatus biofilm metabolism or planktonic A. fumigatus growth significantly less than P. aeruginosa wild type. The common defect of these four mutants was a lack in the production of the P. aeruginosa siderophore pyoverdine. Pure pyoverdine affected A. fumigatus biofilm metabolism, and restored inhibition by the above mutants. In lungs from cystic fibrosis patients, pyoverdine production and antifungal activity correlated. The key inhibitory mechanism for pyoverdine was iron-chelation and denial of iron to A. fumigatus. Further experiments revealed a counteracting, self-protective mechanism by A. fumigatus, based on A. fumigatus siderophore production.

Studies of Pseudomonas aeruginosa Mutants Indicate Pyoverdine as the Central Factor in Inhibition of Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatusin vitro We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatusIMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.

Iron: an essential nutrient for Aspergillus fumigatus and a fulcrum for pathogenesis.

PURPOSE OF REVIEW: Aspergillus fumigatus is a ubiquitous saprophytic fungus that can cause life-threatening invasive aspergillosis in immunocompromised patients. Apart from the immune status of the host only a few characterized virulence factors have been identified. In this review, we describe the role of iron in the manifestation of A. fumigatus virulence. RECENT FINDINGS: We gathered recent clinical evidence suggesting that tissue iron overload increases the risk of invasive aspergillosis occurrence. Furthermore, we summarize the mechanisms that A. fumigatus employs to achieve iron homeostasis and their importance in A. fumigatus proliferation in vitro. We describe two recent in-vivo models that clearly demonstrate the importance of iron in A. fumigatus growth and invasion. SUMMARY: Based on these recent findings, therapy aimed at managing A. fumigatus iron homeostasis locally could make conditions more favorable to the host.

Small Colony Variants of Pseudomonas aeruginosa Display Heterogeneity in Inhibiting Aspergillus fumigatus Biofilm.

Pseudomonas aeruginosa and Aspergillus fumigatus are major microbes in cystic fibrosis (CF). We reported non-mucoid P. aeruginosa isolates more inhibitory to A. fumigatus than mucoid ones. Another CF P. aeruginosa phenotype, small colony variants (SCVs), is an unknown factor in intermicrobial competition with A. fumigatus. Clinical SCV isolates and reference CF non-mucoid isolate (Pa10, producing normal-sized colonies) were compared. Live cells of P. aeruginosa or filtrates from P. aeruginosa planktonic or biofilm cultures were co-incubated with A. fumigatus growing under conditions allowing biofilm formation or with preformed biofilm. Metabolic activity of A. fumigatus biofilm was then measured. When necessary, assays were done after adjustment for growth differences by adding fresh medium to the planktonic culture filtrate. Pyoverdine determinations were performed spectrophotometrically on the planktonic culture filtrates. In all experimental conditions (live cells and planktonic or biofilm culture filtrates of P. aeruginosa versus A. fumigatus biofilm formation or preformed biofilm), three SCV isolates were less inhibitory than Pa10, two equal or more inhibitory. Adjusting planktonic culture filtrates for growth differences showed SCV inhibition differences variably related to growth or deficient inhibitor production. Studies suggested the principal P. aeruginosa inhibitor to be pyoverdine. SCV isolates appear heterogeneous in their capacity to inhibit A. fumigatus biofilm. SCV isolates can be important in the CF microbiome, because they are capable of intermicrobial inhibition.

Pseudomonas phage inhibition of Candida albicans.

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

Haem oxygenase-1 polymorphisms can affect HCV replication and treatment responses with different efficacy in humanized mice.

BACKGROUND & AIMS: Enhancement of host anti-oxidant enzymes, such as haemoxygenase-1, may attenuate virus-mediated hepatocyte injury, while the induction of HO-1 by cobalt-protoporphyrin-IX (CoPP) administration, as the application of its haem degradation product biliverdin (BV), was shown to hinder HCV replication in vitro. In addition, (GT)n -repeats length in the polymorphic region of the HO-1 promoter may affect HO-1 expression and responsiveness to infection and disease severity. Aim of this study was to investigate the antiviral and hepatoprotective effects of CoPP-mediated HO-1 induction, alone or in combination with interferon alpha (peg-IFNα), in HCV-infected mice harbouring hepatocytes from donors with different HO-1-promoter polymorphisms. METHODS: Upon establishment of HCV infection, CoPP, BV and peg-IFNα were given alone or in combination. Viraemia changes and intrahepatic human gene expression were determined by qRT-PCR and immunohistochemistry. RESULTS: CoPP administration increased human HO-1 expression and significantly reduced viraemia, although changes correlated with promoter length (Δ0.5log and Δ2log reduction with medium- and short-polymorphism respectively). Polymorphisms did not influence BV-mediated antiviral effects (Δ1log). Notably, HO-1 induction attenuated basal HCV-driven enhancement of interferon genes and pro-inflammatory cytokines, both in cells with short- or medium-polymorphisms. Moreover, simultaneous administration of CoPP and peg-IFNα reduced viraemia even stronger (median 3log), whereas 1log viraemia reduction was determined in mice receiving peg-IFNα monotherapy. CONCLUSIONS: Although the protective function of HO-1 could be elicited in vivo with both host polymorphisms, the strength of HO-1 induction and suppression of HCV occurred in a polymorphism-dependent manner, indicating that host-genetic determinants may affect disease progression and infection outcome.

Khaya grandifoliola C.DC: a potential source of active ingredients against hepatitis C virus in vitro.

In this study, we examined the antiviral properties of Khaya grandifoliola C.DC (Meliaceae) on the hepatitis C virus (HCV) life cycle in vitro and identified some of the chemical constituents contained in the fraction with the most antiviral activity. Dried bark powder was extracted by maceration in a methylene chloride/methanol (MCM) system (50:50; v/v) and separated on silica gel by flash chromatography. Infection and replication rates in Huh-7 cells were investigated by luciferase reporter assay and indirect immunofluorescence assay using subgenomic replicons, HCV pseudotyped particles, and cell-culture-derived HCV (HCVcc), respectively. Cell viability was assessed by MTT assay, and cellular gene expression was analysed by qRT-PCR. The chemical composition of the fraction with the most antiviral activity was analysed by coupled gas chromatography and mass spectrometry (GC-MS). Five fractions of different polarities (F0-F100) were obtained from the MCM extract. One fraction (KgF25) showed the strongest antiviral effect on LucUbiNeoET replicons at nontoxic concentrations. Tested at 100 µg/mL, KgF25 had a high inhibitory effect on HCV replication, comparable to that of 0.01 µM daclatasvir or 1 µM telaprevir. This fraction also inhibited HCVcc infection by mostly targeting the entry step. KgF25 inhibited HCV entry in a pan-genotypic manner by directly inactivating free viral particles. Its antiviral effects were mediated by the transcriptional upregulation of the haem oxygenase-1 gene and interferon antiviral response. Three constituents, namely, benzene, 1,1'-(oxydiethylidene)bis (1), carbamic acid, (4-methylphenyl)-, 1-phenyl (2), and 6-phenyl, 4-(1'-oxyethylphenyl) hexene (3), were identified from the active fraction KgF25 by GC-MS. Khaya grandifoliola contains ingredients capable of acting on different steps of the HCV life cycle.

Interstrain differences in chronic hepatitis and tumor development in a murine model of inflammation-mediated hepatocarcinogenesis.

UNLABELLED: Chronic inflammation is strongly associated with an increased risk for hepatocellular carcinoma (HCC) development. The multidrug resistance 2 (Mdr2)-knockout (KO) mouse (adenosine triphosphate-binding cassette b4(-/-) ), a model of inflammation-mediated HCC, develops chronic cholestatic hepatitis at an early age and HCC at an adult age. To delineate factors contributing to hepatocarcinogenesis, we compared the severity of early chronic hepatitis and late HCC development in two Mdr2-KO strains: Friend virus B-type/N (FVB) and C57 black 6 (B6). We demonstrated that hepatocarcinogenesis was significantly less efficient in the Mdr2-KO/B6 mice versus the Mdr2-KO/FVB mice; this difference was more prominent in males. Chronic hepatitis in the Mdr2-KO/B6 males was more severe at 1 month of age but was less severe at 3 months of age in comparison with age-matched Mdr2-KO/FVB males. A comparative genome-scale gene expression analysis of male livers of both strains at 3 months of age revealed both common and strain-specific aberrantly expressed genes, including genes associated with the regulation of inflammation, the response to oxidative stress, and lipid metabolism. One of these regulators, galectin-1 (Gal-1), possesses both anti-inflammatory and protumorigenic activities. To study its regulatory role in the liver, we transferred the Gal-1-KO mutation (lectin galactoside-binding soluble 1(-/-) ) from the B6 strain to the FVB strain, and we demonstrated that endogenous Gal-1 protected the liver against concanavalin A-induced hepatitis with the B6 genetic background but not the FVB genetic background. CONCLUSION: Decreased chronic hepatitis in Mdr2-KO/B6 mice at the age of 3 months correlated with a significant retardation of liver tumor development in this strain versus the Mdr2-KO/FVB strain. We found candidate factors that may determine strain-specific differences in the course of chronic hepatitis and HCC development in the Mdr2-KO model, including inefficient anti-inflammatory activity of the endogenous lectin Gal-1 in the FVB strain.

Induction of heme oxygenase 1 prevents progression of liver fibrosis in Mdr2 knockout mice.

UNLABELLED: Induction or overexpression of the heme-degrading enzyme, heme oxygenase 1 (HO-1), has been shown to protect mice from liver damage induced by acute inflammation. We have investigated the effects of HO-1 induction in a mouse model of chronic liver inflammation and fibrogenesis with progression to hepatocellular carcinoma (HCC) (Mdr2ko; FVB.129P2-Abcb4(tm1Bor)). HO-1 was induced in vivo by treatment with cobalt protoporphyrin IX, starting at week 5 or 12 of mice lifespan, and continued for 7 weeks. Our results showed that HO-1 induction reduced liver damage and chronic inflammation by regulating immune cell infiltration or proliferation as well as tumor necrosis factor receptor signaling. Fibrosis progression was significantly reduced by HO-1 induction in mice with mild, as well as established, portal and lobular fibrosis. HO-1 induction significantly suppressed hepatic stellate cell activation. During established fibrosis, HO-1 induction was able to revert portal inflammation and fibrosis below levels observed at the start of treatment. Moreover, hepatocellular proliferation and signs of dysplasia were decreased after HO-1 induction. CONCLUSION: Induction of HO-1 interferes with chronic inflammation and fibrogenesis and, in consequence, might delay progression to HCC.

AfuPmV-1-Infected Aspergillus fumigatus Is More Susceptible to Stress Than Virus-Free Fungus.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) affects Aspergillus fumigatus Af293's growth in vitro, iron metabolism, resistance in intermicrobial competition with Pseudomonas aeruginosa, resistance to osmotic stress, and resistance to the chitin synthase inhibitor nikkomycin Z. Here, we show that response to high temperature, Congo Red-induced stress, and hydrogen peroxide are also dependent on the viral infection status of A. fumigatus. AfuPmV-1- infected Af293 was more susceptible than virus-free Af293 to growth inhibition by high temperature, hydrogen peroxide, Congo Red exposure, and nutrient restriction. Increased resistance of virus-free fungus was observed when cultures were started from conidia but, in the case of high temperature and hydrogen peroxide, not when cultures were started from hyphae. This indicates that the virus impairs the stress response during the growth phase of germination of conidia and development into hyphae. In conclusion, our work indicates that AfuPmV-1 infection in A. fumigatus impairs host responses to stress, as shown by exposure to high temperature, oxidative stress such as hydrogen peroxide, and some cell wall stresses, as shown by exposure to Congo Red (in agreement with our previous observations using nikkomycin Z) and nutrient restriction.

Simple method for quantification of anionic biosurfactants in aqueous solutions.

Biosurfactants are microbial products that have applications as cleaning agents, emulsifiers, and dispersants. Detection and quantification of biosurfactants can be done by various methods, including colorimetric tests, high performance liquid chromatography (HPLC) coupled to several types of detectors, and tests that take advantage of biosurfactants reducing surface tension of aqueous liquids, allowing for spreading and droplet formation of oils. We present a new and simple method for quantifying biosurfactants by their ability, on paper, to reduce surface tension of aqueous solutions, causing droplet dispersion on an oiled surface in correlation with biosurfactant content. We validated this method with rhamnolipids, surfactin, sophorolipids, and ananatoside B; all are anionic microbial surfactants. Linear ranges for quantification in aqueous solutions for all tested biosurfactants were between 10 and 500 µM. Our method showed time-dependent biosurfactant accumulation in cultures of Pseudomonas aeruginosa strains PA14 and PAO1, and Burkholderia thailandensis E264. Mutants in genes responsible for surfactant production showed negligible activity on oiled paper. In summary, our simple assay provides the opportunity to quantify biosurfactant contents of aqueous solutions, for a diversity of surfactants, by means readily available in any laboratory.

Polymycovirus Infection Sensitizes Aspergillus fumigatus for Antifungal Effects of Nikkomycin Z.

Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes (voriconazole, amphotericin) or cell wall glucan synthesis (micafungin, caspofungin). In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94-1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain (MIC 50%, 2 and 4 µg/mL, respectively). Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.