Daniel Bachiller

Sodium colistimethate loaded lipid nanocarriers for the treatment of Pseudomonas aeruginosa infections associated with cystic fibrosis

Lung impairment is the most life-threatening factor for cystic fibrosis patients. Indeed, Pseudomonas aeruginosa is the main pathogen in the pulmonary infection of these patients. In this work, we developed sodium colistimethate loaded lipid nanoparticles, namely, solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC), as a strategy to enhance the antimicrobial therapy against P. aeruginosa in cystic fibrosis patients. The nanoparticles obtained displayed a 200-400 nm size, high drug entrapment (79-94%) and a sustained drug release profile. Moreover, both SLN and NLC presented antimicrobial activity against clinically isolated P. aeruginosa. The integrity of the nanoparticles was not affected by nebulization through a mesh vibrating nebulizer. Moreover, lipid nanoparticles appeared to be less toxic than free sodium colistimethate in cell culture. Finally, an in vivo distribution experiment showed that nanoparticles spread homogenously through the lung and there was no migration of lipid nanoparticles to other organs, such as liver, spleen or kidneys.

Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663–676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949–953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543–549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348–362, 2009), removal of the reprogramming vectors by ‘piggyBac’ transposition Woltjen et al. (Nature 458(7239):766–770, 2009); Kaji et al. (Nature 458(7239):771–775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964–977, 2009), episomal vectors Yu et al. (Science 324(5928):797–801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381–384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376–388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789–797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently.

Killing effect of nanoencapsulated colistin sulfate on Pseudomonas aeruginosa from cystic fibrosis patients

Pseudomonas aeruginosa frequently infects the respiratory tract of cystic fibrosis (CF) patients. Multidrug-resistant phenotypes and high capacity to form stable biofilms are common. Recent studies have described the emergence of colistin-resistant isolates in CF patients treated with long-term inhaled colistin. The use of nanoparticles containing antimicrobials can contribute to overcome drug resistance mechanisms. The aim of this study was to explore antimicrobial activity of nanoencapsulated colistin (SLN-NLC) versus free colistin against P. aeruginosa clinical isolates from CF patients and to investigate their efficacy in biofilm eradication. Susceptibility of planktonic bacteria to antimicrobials was examined by using the broth microdilution method and growth curve assay. Minimal biofilm eradication concentration (MBEC) and biofilm prevention concentration (BPC) were determined to assess antimicrobial susceptibility of sessile bacteria. We used atomic force microscopy (AFM) to visualize treated and untreated biofilms and to determine surface roughness and other relevant parameters. Colistin nanoparticles had the same antimicrobial activity as free drug against planktonic bacteria. However, nanoencapsulated colistin was much more efficient in the eradication of biofilms than free colistin. Thus, these formulations have to be considered as a good alternative therapeutic option to treat P. aeruginosa infections.

Pulmonary delivery of tobramycin-loaded nanostructured lipid carriers for Pseudomonas aeruginosa infections associated with cystic fibrosis.

Among the pathogens that affect cystic fibrosis (CF) patients, Pseudomonas aeruginosa is the most prevalent. As a way to fight against this infection, nanotechnology has emerged over the last decades as a promising alternative to overcome resistance to antibiotics in infectious diseases. The goal of this work was to elaborate and characterize lipid nanoparticles for pulmonary delivery of tobramycin. Tobramycin-loaded nanostructured lipid carriers (Tb-NLCs) were prepared by hot melt homogenization technique. In addition, nanoparticles labeled with infrared dye (IR-NLCs) were used to investigate their in vivo performance after pulmonary administration. Tb-NLCs displayed a mean diameter size around 250 nm, high drug encapsulation (93%) and sustained release profile. Tb-NLCs showed to be active against clinically isolated P. aeruginosa. Moreover, Tb-NLCs did not decrease cell viability and were able to overcome an artificial mucus barrier in the presence of mucolytics agents. During the in vivo assay, IR-NLCs were administered to several mice by the intratracheal route using a Penn Century device. Next, the biodistribution of the nanoparticles was analyzed at different time points showing a wide nanosystem distribution in the lungs. Altogether, tobramycin-loaded NLCs seem to us an encouraging alternative to the currently available CF therapies.

PRODUCTION OF X0 CLONES IN XX FEMALES OF DROSOPHILA

The experiments reported here are aimed at determining whether mutations deleting the function of the Sex-lethal (Sxl) gene are able to suppress the lethality of X0 clones, induced in females after the time when the state of activity of Sxl is irreversibly fixed by the ratio of the number of X chromosomes to sets of autosomes (X:A). This analysis was carried out by comparing the frequency of induced male clones (X0 constitution) in SxlfLS/+ and Sxl+/Sxl+ females, following irradiation at blastoderm and larval stages. The genotype used in these experiments, however, could also give rise to 2X; 2A cells homozygous for SxlfLS, and such cells would also differentiate male structures. To minimize this possibility, we have constructed a genotype made up of a ring and a rod X chromosome. In such ring-rod females the production of 2X; 2A clones homozygous for SxlfLS is a rather rare event, if possible at all. X0 male clones were produced in both types of females following irradiation at blastoderm stage, while X0 male clones were only observed in SxlfLS/+ females when irradiation took place at larval stage. In this latter case, the only X0 male clones were those that contained the SxlfLS mutation. These results support the idea of Sánchez & Nöthiger (1983) that the X:A signal irreversibly sets the state of activity of Sxl at blastoderm stage, and in addition show that X0 clones generated after that time are viable if they contain a Sxl- mutation. These results are compatible with the idea of Sxl being the only gene that responds to the X:A signal.

Further analysis on the male-specific lethal mutations that affect dosage compensation in Drosophila melanogaster

SummaryThe male-specific lethal genes (msl) of D. melanogaster represent a set of genes whose functions are required for the specific X chromosome hypertranscription in males (dosage compensation). We have carried out the clonal analysis of one of those msl mutations: msl-3b. Clones homozygous for msl-3b are deleterious; this mutation presents cell autonomy and in the cases where msl clones appeared in sexually dimorphic regions (5th and 6th tergites) they do not show sexual transformation. Moreover, the lethal phase and the growth dynamics (measured by the protein content during larval growth) are the same for male larvae homozygous for one msl mutation (msl-1) or three msl mutations (msl-2 msl-1 mle), i.e. the msl mutations do not show additive effects. This paper considers the possible interactions between the msl genes that bring about dosage compensation.

Stability study of sodium colistimethate-loaded lipid nanoparticles

Abstract In the last decades, the encapsulation of antibiotics into nanoparticulate carriers has gained increasing attention for the treatment of infectious diseases. Sodium colistimethate-loaded solid lipid nanoparticles (Colist-SLNs) and nanostructured lipid carriers (Colist-NLCs) were designed aiming to treat the pulmonary infection associated to cystic fibrosis patients. The nanoparticles were freeze-dried using trehalose as cryoprotectant. The stability of both nanoparticles was analysed over one year according to the International Conference of Harmonisation (ICH) guidelines by determining the minimum inhibitory concentration (MIC) against clinically isolated Pseudomonas aeruginosa strains and by studying their physico-chemical characteristics. The results showed that Colist-SLNs lost their antimicrobial activity at the third month; on the contrary, the antibacterial activity of Colist-NLCs was maintained throughout the study within an adequate range (MIC ≤16 μg/mL). In addition, Colist-NLCs exhibited suitable physico-chemical properties at 5 °C and 25 °C/60% relative humidity over one year. Altogether, Colist-NLCs proved to have better stability than Colist-SLNs.

Tracheal oxalosis associated with Aspergillus niger tracheobronchitis

To the Editor: Aspergillus is a widespread mould that can cause a variety of human diseases, usually in the setting of immunosuppression. Invasive pulmonary aspergillosis is the most aggressive form of Aspergillus infection and it is associated with morbidity and mortality. Invasive Aspergillus tracheobronchitis is a rare entity, primarily affecting lung transplant recipients, and patients with AIDS or chronic obstructive pulmonary disease (COPD). Aspergillus niger , and to a lesser extent A. fumigatus , can cause calcium oxalate crystals to accumulate in the lung, a condition termed pulmonary oxalosis. Tracheal oxalosis due to invasive Aspergillus tracheobronchitis has, to our knowledge, not been described before. Here, we report the case of a patient with probable invasive Aspergillus tracheobronchitis who developed tracheal oxalosis. Clinical, radiological and pathological correlation is given. Invasive pulmonary aspergillosis is the most aggressive form of Aspergillus infection and it is related with a high morbidity and mortality [1]. The majority of patients with invasive aspergillosis are critically ill, requiring intensive care unit (ICU) admission. The presence of Aspergillus in the airways of critically ill patients carries higher mortality risk [2], and should prompt further diagnostic evaluation, including fibre-optic bronchoscopy [3]. Invasive Aspergillus tracheobronchitis is thought to be a rare entity, primarily …

Generation of two induced pluripotent stem cells lines from Mucopolysaccharydosis IIIA patient: IMEDEAi004-A and IMEDEAi004-B

Mucoplysaccharydosis IIIA (MPSIIIA) is the most severe form of Sanfilippo syndrome. Skin fibroblasts from a MPSIIIA compound heterozygous (E447K/R245H) patient were nucleofected with four OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-Myc, LIN28, BCL-xL and shp53. The two iPSCs lines generated carry both sulfamidase enzyme (SGSH) mutations, are free of plasmid integration, have normal karyotype, express pluripotency-associated markers and are able to differentiate into the three germ layers.

Generation of two induced pluripotent stem cell (iPSC) lines from p.F508del Cystic Fibrosis patients

Cystic Fibrosis (CF) is a monogenic, lethal disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) gene. Here we report the production of CF-iPS cell lines from two different p.F508del homozygous female patients (Table 1). Two different primary cell types, skin fibroblasts and keratinocytes, were transfected with retroviral cocktails containing four: c-MYC, KLF4, OCT4 and SOX2 (MKOS) or three: KLF4, OCT4 and SOX2 (KOS) reprogramming factors. Two fibroblast-derived MKOS lines are described in the main text. The lines carry the p.F508del mutation, have a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.