Gloria Molero - Academia.edu (original) (raw)

Papers by Gloria Molero

Antioxidants, Apr 26, 2024

Infection and Immunity, Feb 1, 1997

Deletion of the Candida albicans mitogen-activated protein kinase MKC1 gene gave rise to viable c... more Deletion of the Candida albicans mitogen-activated protein kinase MKC1 gene gave rise to viable cells whose cell integrity was affected (F. Navarro-García, M.

Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Mar 8, 2007

Journal of Bacteriology, May 15, 1999

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was address... more The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.

Journal of Proteomics, 2021

Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial ... more Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under very different conditions and from diverse cellular compartments have been analyzed and joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1. This is the highest published coverage among the eight bacterial PeptideAtlas datasets currently available. The proteins in the Pseudomonas PeptideAtlas cover 84% of metabolic proteins, 71% of proteins involved in genetic information processing, 72% of proteins responsible for environmental information processing, more than 80% of proteins related to quorum sensing and biofilm formation, and 81% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. Here we describe how this resource was built and some of the physiologically important proteins of this pathogen. Significance Pseudomonas aeruginosa is among the most versatile bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

Access Microbiology, 2021

Fungal infections are a global health problem. Of them, those produced by Candida albicans are th... more Fungal infections are a global health problem. Of them, those produced by Candida albicans are the most important, with a reduced arsenal of antifungals and an increasing problem of antifungal resistance. Thus, the discovery of new antifungal targets and drugs remains interesting. Metformin is a biguanide administered as a first-line treatment for Type II Diabetes Mellitus and it has recently been published its anti-Candida action, especially against C. glabrata, and its synergistic effect with other antifungals. Our studies of the effect of metformin on C. albicans have revealed an inhibition of growth, filamentation and other phenotypes important for virulence. Although metformin has been described as an AMPK agonist, its mechanism of action is partly unknown. To deepen into the anti-Candida mechanism of action, we have addressed the differential proteomic study. A set-up of the conditions for the proteomic study has been carried out, fixing a concentration of 50mM of metformin, 6...

International Journal of Pharmaceutics, 2015

The aim of this work is to study the micelle systems of amphotericin B (AmB) and surfactant sodiu... more The aim of this work is to study the micelle systems of amphotericin B (AmB) and surfactant sodium deoxycholate (NaDC) as possible formulations to treat brain fungal infections. Fungizone(®) and Ambisome(®) were used as AmB references. The particle size, aggregation state, toxicity and efficacy of AmB:NaDC micelles were studied with increasing proportions of NaDC. Differences in the size and aggregation state of the reference formulations and micellar NaDC formulations might explain the differences in their distribution and therefore in their toxicity and efficacy. AmB:NaDC 1:0.8 and 1:1.5 nano-sized micelle systems showed a poly-aggregated form of AmB and small mean particle size (450-750 nm). The AmB:NaDC 1:0.8 and AmB:NaDC 1:1.5 micelle systems studied showed an 8-fold lower toxicity than Fungizone(®). Efficacy was examined in a murine candidiasis model by determining the survival rate and tissue burden reduction in kidneys and brain. The AmB:NaDC 1:1.5 micellar system at 5mg/kg of AmB and the highest amount of NaDC (7.5 mg/kg) presented a good survival rate, and induced a major clearance of brain infection. The new AmB:NaDC 1:1.5 nano-sized micelle system is a promising formulation with a good efficacy/toxicity ratio, which can be attributed to its particle size, AmB aggregation state and NaDC content.

International Journal of Food Microbiology, 2011

In vivo virulence Baker's strain S. cerevisiae var. boulardii Two commercial Saccharomyces cerevi... more In vivo virulence Baker's strain S. cerevisiae var. boulardii Two commercial Saccharomyces cerevisiae strains, a baker's strain and the bio-therapeutic agent Ultralevure, have been proposed as a possible exogenous source of human colonization (de Llanos et al., 2004, 2006a). Moreover, these strains express phenotypical traits associated to pathogenicity (de Llanos et al., 2006b). Taking into account that both commercial preparations represent an important source of living S. cerevisiae cells we have performed an in vivo study to evaluate whether there is a potential safety risk to humans. Their virulence was compared with that of other commercial strains with less virulent traits, and with clinical isolates, using two murine models (BALB/c and DBA/2N mice). Burden determination in the brain and kidneys showed that the ability to disseminate, colonize and persist was manifested not only by clinical isolates but also by commercial strains. Among these, the baker's strain and Ultralevure were able to cause the death of BALB/c mice at rates similar to those shown by two of the clinical isolates. These results highlight the pathogenic potential of these strains and show that four-week-old BALB/c mice are an appropriate murine model to study the virulence of yeasts with low or moderate pathogenicity. Furthermore, we have shown the positive effect of an immunosuppressive therapy with cyclophosphamide in the virulence of the baker's strains and Ultralevure but not in the rest of the commercial strains under study. The data suggest that although S. cerevisiae has always been considered a GRAS microorganism, commercial preparations should include only those strains shown to be safe in order to minimize complications in risk groups.

International Journal of Antimicrobial Agents, 2008

A new poly-aggregated form of amphotericin B was formulated as a non-microencapsulated form (P-AM... more A new poly-aggregated form of amphotericin B was formulated as a non-microencapsulated form (P-AMB) or incorporated in albumin microspheres (MP-AMB) and compared with the conventional amphotericin B formulation (D-AMB). Mice were infected with Candida albicans and treated with two different intermittent dose regimens of the different amphotericin B formulations. Efficacy and toxicity were studied by the determination of survival rate, kidney colony-forming units counts, biochemical parameters and amphotericin B concentrations in plasma and organs. All the treatments significantly (P < 0.05) increased the survival rate in relation to the untreated group, although nonstatistically significant differences (P > 0.05) were found between formulations and dosing regimens. All the treatments produced kidney toxicity, expressed by high urea levels. Kidney toxicity was especially significant for mice treated with the D-AMB formulation where unilateral kidney atrophy was observed in most of the mice, whereas most of the mice treated with P-AMB conserved both kidneys with a normal size and appearance. At 45 days post infection, variable distribution of amphotericin B in the body was obtained depending on the amphotericin B formulation. In conclusion, non-daily dosing regimens of P-AMB, which is less toxic than D-AMB, could be used as an alternative to the conventional D-AMB formulation to treat experimental candidiasis.

International Journal of Pharmaceutics, 2012

Amphotericin B (AmB) is a wide spectrum antifungal with low incidence of clinical resistance. How... more Amphotericin B (AmB) is a wide spectrum antifungal with low incidence of clinical resistance. However, there are no licensed topical formulations with AmB in most developed countries. Extemporaneous preparations of AmB are frequently prepared from available marketed parenteral formulations. Herein, a solution of AmB with ␥-cyclodextrin is described as suitable for topical administration as eye drops. This novel formulation is characterised by its ability to solubilise AmB and to maintain its antifungal activity, physicochemical stability and sterility over 30 days. Antifungal activity against Candida albicans was significantly higher (35%) for the new formulation than that obtained with Fungizone ® based extemporaneous prepared suspension. Optimal 0.1% AmB-10% cyclodextrin formulation remained sterile and with an acceptable osmolarity, pH and particle size for ophthalmic use over 4 weeks. Complexation with ␥-cyclodextrins improved AmB chemical stability compared to the reference eye drops suspension based on Fungizone ®. These results illustrate the feasibility of an ophthalmic AmB formulation easy enough to be licensed or prepared in community and hospital pharmacies.

International Journal of Pharmaceutics, 2014

Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of cli... more Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of clinical resistance. Its parenteral administration has high risk of nephrotoxicity that limits its use. In order to treat cutaneous infections, AmB topical administration is a safer therapy because of the low systemic absorption of the drug across mucous membranes. Moreover, in some developing countries both fungal topical infections and cutaneous leishmaniasis are an important health problem. The aim of this work is to formulate a topical amphotericin preparation and test its in vitro antifungal (against 11 different fungal species) and antileishmanial activity. γ-Cyclodextrin (γ-CD) was chosen to solubilise AmB. Furthermore, γ-CD has shown a synergistic effect on membrane destabilization with AmB. Topical novel formulations based on AmB-CD complex have exhibited greater antifungal activity (48%, 28% and 60% higher) when compared to AmB Neo-Sensitabs(®) disks, AmB dissolved in dimethyl sulfoxide (DMSO) and Clotrimazole(®) cream, respectively. Furthermore, AmB-CD methyl cellulose gel has shown significantly higher inhibition activity on biofilm formation, larger penetration through yeast biofilms and higher fungicidal activity on biofilm cells compared to AmB dissolved in DMSO. In addition, AmB-CD gel exhibited both high in vitro leishmanicidal efficacy with wider therapeutic index (between 2 and 8-fold higher than AmB deoxycholate depending on Leishmania spp.) and also in vivo activity in an experimental model of cutaneous leishmaniasis. These results illustrate the feasibility of a topical AmB formulation easy to prepare, physicochemically stable over 6 months, safe and effective against diverse fungal and parasitic cutaneous infections.

The Journal of Cell Biology, 1998

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15... more The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells...

Journal of Antimicrobial Chemotherapy, 2008

The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic ... more The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB). Methods: The therapeutic efficacy and toxicity of amphotericin B formulations was studied in an immunocompetent murine model of systemic candidiasis. A pharmacokinetic study was also performed to measure the plasma, kidney, liver and spleen amphotericin B concentrations after administration of the three formulations to mice. Results: The acute toxicity of P-AMB in mice is lower than that of the conventional amphotericin B reference formulation (D-AMB). The 50% lethal doses were increased at least eight times. Intravenous bolus administration of doses up to 40 mg/kg of body weight of poly-aggregated amphotericin B, either P-AMB or MP-AMB, did not produce acute symptoms of toxicity. Interestingly, in the pharmacokinetic study, significant (P < 0.05) lower plasma and kidney amphotericin B concentrations and higher liver and spleen amphotericin B concentrations were achieved after poly-aggregated amphotericin B formulation (P-AMB and MP-AMB) administration in relation to the reference formulation (D-AMB). At high amphotericin B doses, no significant differences in efficacy (P > 0.05) were observed among the formulations (D-AMB, P-AMB and MP-AMB). Conclusions: Although the efficacy in the candidiasis treatment was decreased as a consequence of amphotericin B aggregation, it can be compensated by the possibility of increasing the doses with lower nephrotoxicity. Moreover, due to its lower toxicity while maintaining its effectiveness, the polyaggregated formulations (P-AMB and MP-AMB) have a better therapeutic index than the conventional formulation (D-AMB).

Journal of Bacteriology, 1993

LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detaile... more LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detailed characterization of mutants carrying the lyt1-1 allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions. At the nonpermissive temperature of 37 degrees C, lyt1 haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells. These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis. Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly. On the other hand, lyt1-1/lyt1-1 diploids failed to sporulate at both 24 and 37 degrees C. Taking into account these characteri...

FEMS Microbiology Letters, 1999

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cere... more The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37³C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.

... P. Thomas, José Luis López-Ribot, César Nombela, and Concha Gil Abstract Proteomics may signi... more ... P. Thomas, José Luis López-Ribot, César Nombela, and Concha Gil Abstract Proteomics may significantly contribute towards a better ... yale. edu/intmed/infdis/candida, which was defined computationally by using computer-based prediction algorithms and C. albi-cans genomic ...

Journal of Antimicrobial Chemotherapy, 2008

Objectives: The purpose of this investigation is the study of toxicity, in vivo distribution and ... more Objectives: The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB).

Journal of Proteome Research, 2016

The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effecti... more The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, non-coding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus, we have undertaken the first quantitative proteomic analysis on the protein composition of THP1 macrophages-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP1 non-differenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.

Antioxidants, Apr 26, 2024

Infection and Immunity, Feb 1, 1997

Deletion of the Candida albicans mitogen-activated protein kinase MKC1 gene gave rise to viable c... more Deletion of the Candida albicans mitogen-activated protein kinase MKC1 gene gave rise to viable cells whose cell integrity was affected (F. Navarro-García, M.

Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Mar 8, 2007

Journal of Bacteriology, May 15, 1999

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was address... more The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.

Journal of Proteomics, 2021

Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial ... more Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under very different conditions and from diverse cellular compartments have been analyzed and joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1. This is the highest published coverage among the eight bacterial PeptideAtlas datasets currently available. The proteins in the Pseudomonas PeptideAtlas cover 84% of metabolic proteins, 71% of proteins involved in genetic information processing, 72% of proteins responsible for environmental information processing, more than 80% of proteins related to quorum sensing and biofilm formation, and 81% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. Here we describe how this resource was built and some of the physiologically important proteins of this pathogen. Significance Pseudomonas aeruginosa is among the most versatile bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

Access Microbiology, 2021

Fungal infections are a global health problem. Of them, those produced by Candida albicans are th... more Fungal infections are a global health problem. Of them, those produced by Candida albicans are the most important, with a reduced arsenal of antifungals and an increasing problem of antifungal resistance. Thus, the discovery of new antifungal targets and drugs remains interesting. Metformin is a biguanide administered as a first-line treatment for Type II Diabetes Mellitus and it has recently been published its anti-Candida action, especially against C. glabrata, and its synergistic effect with other antifungals. Our studies of the effect of metformin on C. albicans have revealed an inhibition of growth, filamentation and other phenotypes important for virulence. Although metformin has been described as an AMPK agonist, its mechanism of action is partly unknown. To deepen into the anti-Candida mechanism of action, we have addressed the differential proteomic study. A set-up of the conditions for the proteomic study has been carried out, fixing a concentration of 50mM of metformin, 6...

International Journal of Pharmaceutics, 2015

The aim of this work is to study the micelle systems of amphotericin B (AmB) and surfactant sodiu... more The aim of this work is to study the micelle systems of amphotericin B (AmB) and surfactant sodium deoxycholate (NaDC) as possible formulations to treat brain fungal infections. Fungizone(®) and Ambisome(®) were used as AmB references. The particle size, aggregation state, toxicity and efficacy of AmB:NaDC micelles were studied with increasing proportions of NaDC. Differences in the size and aggregation state of the reference formulations and micellar NaDC formulations might explain the differences in their distribution and therefore in their toxicity and efficacy. AmB:NaDC 1:0.8 and 1:1.5 nano-sized micelle systems showed a poly-aggregated form of AmB and small mean particle size (450-750 nm). The AmB:NaDC 1:0.8 and AmB:NaDC 1:1.5 micelle systems studied showed an 8-fold lower toxicity than Fungizone(®). Efficacy was examined in a murine candidiasis model by determining the survival rate and tissue burden reduction in kidneys and brain. The AmB:NaDC 1:1.5 micellar system at 5mg/kg of AmB and the highest amount of NaDC (7.5 mg/kg) presented a good survival rate, and induced a major clearance of brain infection. The new AmB:NaDC 1:1.5 nano-sized micelle system is a promising formulation with a good efficacy/toxicity ratio, which can be attributed to its particle size, AmB aggregation state and NaDC content.

International Journal of Food Microbiology, 2011

In vivo virulence Baker's strain S. cerevisiae var. boulardii Two commercial Saccharomyces cerevi... more In vivo virulence Baker's strain S. cerevisiae var. boulardii Two commercial Saccharomyces cerevisiae strains, a baker's strain and the bio-therapeutic agent Ultralevure, have been proposed as a possible exogenous source of human colonization (de Llanos et al., 2004, 2006a). Moreover, these strains express phenotypical traits associated to pathogenicity (de Llanos et al., 2006b). Taking into account that both commercial preparations represent an important source of living S. cerevisiae cells we have performed an in vivo study to evaluate whether there is a potential safety risk to humans. Their virulence was compared with that of other commercial strains with less virulent traits, and with clinical isolates, using two murine models (BALB/c and DBA/2N mice). Burden determination in the brain and kidneys showed that the ability to disseminate, colonize and persist was manifested not only by clinical isolates but also by commercial strains. Among these, the baker's strain and Ultralevure were able to cause the death of BALB/c mice at rates similar to those shown by two of the clinical isolates. These results highlight the pathogenic potential of these strains and show that four-week-old BALB/c mice are an appropriate murine model to study the virulence of yeasts with low or moderate pathogenicity. Furthermore, we have shown the positive effect of an immunosuppressive therapy with cyclophosphamide in the virulence of the baker's strains and Ultralevure but not in the rest of the commercial strains under study. The data suggest that although S. cerevisiae has always been considered a GRAS microorganism, commercial preparations should include only those strains shown to be safe in order to minimize complications in risk groups.

International Journal of Antimicrobial Agents, 2008

A new poly-aggregated form of amphotericin B was formulated as a non-microencapsulated form (P-AM... more A new poly-aggregated form of amphotericin B was formulated as a non-microencapsulated form (P-AMB) or incorporated in albumin microspheres (MP-AMB) and compared with the conventional amphotericin B formulation (D-AMB). Mice were infected with Candida albicans and treated with two different intermittent dose regimens of the different amphotericin B formulations. Efficacy and toxicity were studied by the determination of survival rate, kidney colony-forming units counts, biochemical parameters and amphotericin B concentrations in plasma and organs. All the treatments significantly (P < 0.05) increased the survival rate in relation to the untreated group, although nonstatistically significant differences (P > 0.05) were found between formulations and dosing regimens. All the treatments produced kidney toxicity, expressed by high urea levels. Kidney toxicity was especially significant for mice treated with the D-AMB formulation where unilateral kidney atrophy was observed in most of the mice, whereas most of the mice treated with P-AMB conserved both kidneys with a normal size and appearance. At 45 days post infection, variable distribution of amphotericin B in the body was obtained depending on the amphotericin B formulation. In conclusion, non-daily dosing regimens of P-AMB, which is less toxic than D-AMB, could be used as an alternative to the conventional D-AMB formulation to treat experimental candidiasis.

International Journal of Pharmaceutics, 2012

Amphotericin B (AmB) is a wide spectrum antifungal with low incidence of clinical resistance. How... more Amphotericin B (AmB) is a wide spectrum antifungal with low incidence of clinical resistance. However, there are no licensed topical formulations with AmB in most developed countries. Extemporaneous preparations of AmB are frequently prepared from available marketed parenteral formulations. Herein, a solution of AmB with ␥-cyclodextrin is described as suitable for topical administration as eye drops. This novel formulation is characterised by its ability to solubilise AmB and to maintain its antifungal activity, physicochemical stability and sterility over 30 days. Antifungal activity against Candida albicans was significantly higher (35%) for the new formulation than that obtained with Fungizone ® based extemporaneous prepared suspension. Optimal 0.1% AmB-10% cyclodextrin formulation remained sterile and with an acceptable osmolarity, pH and particle size for ophthalmic use over 4 weeks. Complexation with ␥-cyclodextrins improved AmB chemical stability compared to the reference eye drops suspension based on Fungizone ®. These results illustrate the feasibility of an ophthalmic AmB formulation easy enough to be licensed or prepared in community and hospital pharmacies.

International Journal of Pharmaceutics, 2014

Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of cli... more Amphotericin B (AmB) has a broad antifungal and leishmanicidal activity with low incidence of clinical resistance. Its parenteral administration has high risk of nephrotoxicity that limits its use. In order to treat cutaneous infections, AmB topical administration is a safer therapy because of the low systemic absorption of the drug across mucous membranes. Moreover, in some developing countries both fungal topical infections and cutaneous leishmaniasis are an important health problem. The aim of this work is to formulate a topical amphotericin preparation and test its in vitro antifungal (against 11 different fungal species) and antileishmanial activity. γ-Cyclodextrin (γ-CD) was chosen to solubilise AmB. Furthermore, γ-CD has shown a synergistic effect on membrane destabilization with AmB. Topical novel formulations based on AmB-CD complex have exhibited greater antifungal activity (48%, 28% and 60% higher) when compared to AmB Neo-Sensitabs(®) disks, AmB dissolved in dimethyl sulfoxide (DMSO) and Clotrimazole(®) cream, respectively. Furthermore, AmB-CD methyl cellulose gel has shown significantly higher inhibition activity on biofilm formation, larger penetration through yeast biofilms and higher fungicidal activity on biofilm cells compared to AmB dissolved in DMSO. In addition, AmB-CD gel exhibited both high in vitro leishmanicidal efficacy with wider therapeutic index (between 2 and 8-fold higher than AmB deoxycholate depending on Leishmania spp.) and also in vivo activity in an experimental model of cutaneous leishmaniasis. These results illustrate the feasibility of a topical AmB formulation easy to prepare, physicochemically stable over 6 months, safe and effective against diverse fungal and parasitic cutaneous infections.

The Journal of Cell Biology, 1998

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15... more The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells...

Journal of Antimicrobial Chemotherapy, 2008

The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic ... more The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB). Methods: The therapeutic efficacy and toxicity of amphotericin B formulations was studied in an immunocompetent murine model of systemic candidiasis. A pharmacokinetic study was also performed to measure the plasma, kidney, liver and spleen amphotericin B concentrations after administration of the three formulations to mice. Results: The acute toxicity of P-AMB in mice is lower than that of the conventional amphotericin B reference formulation (D-AMB). The 50% lethal doses were increased at least eight times. Intravenous bolus administration of doses up to 40 mg/kg of body weight of poly-aggregated amphotericin B, either P-AMB or MP-AMB, did not produce acute symptoms of toxicity. Interestingly, in the pharmacokinetic study, significant (P < 0.05) lower plasma and kidney amphotericin B concentrations and higher liver and spleen amphotericin B concentrations were achieved after poly-aggregated amphotericin B formulation (P-AMB and MP-AMB) administration in relation to the reference formulation (D-AMB). At high amphotericin B doses, no significant differences in efficacy (P > 0.05) were observed among the formulations (D-AMB, P-AMB and MP-AMB). Conclusions: Although the efficacy in the candidiasis treatment was decreased as a consequence of amphotericin B aggregation, it can be compensated by the possibility of increasing the doses with lower nephrotoxicity. Moreover, due to its lower toxicity while maintaining its effectiveness, the polyaggregated formulations (P-AMB and MP-AMB) have a better therapeutic index than the conventional formulation (D-AMB).

Journal of Bacteriology, 1993

LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detaile... more LYT1 is an essential gene for the growth and morphogenesis of Saccharomyces cerevisiae. A detailed characterization of mutants carrying the lyt1-1 allele showed that this mutation was recessive and pleiotropic, affecting both mitotic and meiotic functions. At the nonpermissive temperature of 37 degrees C, lyt1 haploid strains budded at a distal position (instead of an axial one, as in wild-type haploid strains) and underwent autolysis when the buds were almost the size of the mother cells. These mitotic alterations in cell stability and budding topology were dependent on growth and protein synthesis. Autolysis was prevented by inhibiting DNA synthesis (with hydroxyurea) or by blocking the assembly of microtubules (with benomyl), suggesting that loss of cell viability must occur at a fixed mitotic cycle stage after DNA synthesis and mitotic spindle assembly. On the other hand, lyt1-1/lyt1-1 diploids failed to sporulate at both 24 and 37 degrees C. Taking into account these characteri...

FEMS Microbiology Letters, 1999

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cere... more The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37³C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.

... P. Thomas, José Luis López-Ribot, César Nombela, and Concha Gil Abstract Proteomics may signi... more ... P. Thomas, José Luis López-Ribot, César Nombela, and Concha Gil Abstract Proteomics may significantly contribute towards a better ... yale. edu/intmed/infdis/candida, which was defined computationally by using computer-based prediction algorithms and C. albi-cans genomic ...

Journal of Antimicrobial Chemotherapy, 2008

Objectives: The purpose of this investigation is the study of toxicity, in vivo distribution and ... more Objectives: The purpose of this investigation is the study of toxicity, in vivo distribution and therapeutic activity against candidiasis of poly-aggregated amphotericin B, in two different formulations: not microencapsulated (P-AMB) or incorporated in albumin microspheres (MP-AMB).

Journal of Proteome Research, 2016

The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effecti... more The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, non-coding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus, we have undertaken the first quantitative proteomic analysis on the protein composition of THP1 macrophages-derived EVs during the interaction with Candida albicans. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP1-derived EVs, both from control and from Candida-infected macrophages, had similar effector functions on other THP1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP1 non-differenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to C. albicans infection and in macrophages communication.