Gamazo, C. (Carlos)
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- Novel bioactive hydrophobic gentamicin carriers for the treatment of intracellular bacterial infections.(Elsevier, 2011) Veciana, J. (Jaume); Elizondo, E. (Elisa); Blanco-Prieto, M.J. (María José); Gamazo, C. (Carlos); Moreno-Calvo, E. (Evelyn); Imbuluzqueta, E. (Edurne); Ventosa, N. (Nora)Gentamicin (GEN) is an aminoglycoside antibiotic with a potent antibacterial activity against a wide variety of bacteria. However, its poor cellular penetration limits its use in the treatment of infections caused by intracellular pathogens. One potential strategy to overcome this problem is the use of particulate carriers that can target the intracellular sites of infection. In this study GEN was ion paired with the anionic AOT surfactant to obtain a hydrophobic complex (GEN-AOT) that was formulated as a particulated material either by the Precipitation with a Compressed Antisolvent (PCA) method, or by encapsulation into poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). The micronization of GEN-AOT by PCA yielded a particulated material with a higher surface area than the non-precipitated complex, while PLGA NPs within a size range of 250-330 nm and a sustained release of the drug over 70 days were obtained by preparing the NPs using the emulsion solvent evaporation method. For the first time, GEN encapsulation efficiency values around 100% were achieved for the different NP formulations with no signs of interaction between the drug and the polymer. Finally, in vitro studies against the intracellular bacteria Brucella melitensis, used as a model of intracellular pathogen, demonstrated that the bactericidal activity of GEN was unmodified after ion-pairing, precipitation or encapsulation into NPs. These results, encourage their use for treatment for infections caused by GEN sensitive intracellular bacteria.
- Gamma interferon loaded onto albumin nanoparticles: in vitro and in vivo activities against Brucella abortus(American Society of Microbiology, 2007) Espuelas, S. (Socorro); Gamazo, C. (Carlos); Irache, J.M. (Juan Manuel); Segura, S. (S.)The aim of this study was to evaluate the activity of gamma interferon (IFN-gamma) when it was either adsorbed onto or loaded into albumin nanoparticles. Brucella abortus-infected macrophages and infected BALB/c mice were selected as the models for testing of the therapeutic potentials of these cytokine delivery systems, in view of the well-established role of IFN-gamma-activated macrophages for the control of Brucella sp. infections. Whereas the encapsulation of IFN-gamma inside the matrix of nanoparticles completely abrogated its activity, adsorbed IFN-gamma increased by 0.75 log unit the bactericidal effect induced by RAW macrophages activated with free IFN-gamma, along with a higher level of production of nitric oxide. In infected BALB/c-mice, IFN-gamma adsorbed onto nanoparticles was also more active than free cytokine in reducing the number of bacteria in the spleens, and the effect was mediated by an increased ratio of IFN-gamma-secreting (Th1) to interleukin-4-secreting (Th2) cells. Overall, albumin nanoparticles would be suitable as carriers that target IFN-gamma to macrophages and, thus, potentiate their therapeutic activity.
- Protective passive immunity in escherichia coli ETEC-challenged neonatal mice conferred by orally immunized dams with nanoparticles containing homologous outer membrane vesicles(MDPI AG, 2020) Gamazo, C. (Carlos); Pastor, Y. (Yadira); Matías, J. (Jose); Irache, J.M. (Juan Manuel)Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in mammals, including neonatal, recently weaned pigs and infant human beings. We have previously shown that outer membrane vesicles (OMV) obtained from ETEC serotypes encapsulated into zein nanoparticles, coated with a Gantrez-mannosamine polymer conjugate (OMV-NP), were immunogenic in mice and sows. In the present study, we show that pups from vaccinated mice were protected against ETEC F4 serotype challenge through maternal passive immunization. OMV from F4 cultures were collected and characterized. Two-week-pregnant BALB/c mice were orally immunized with a single dose of vesicles (0.2 mg) either free (OMV) or encapsulated into nanoparticles (OMV-NP). Evaluation of the antibodies in serum (IgG1, Ig2a or IgA) and feces (IgA) of dams immunized with OMV-NP revealed an enhancement of specific immunogenicity. The antibody response conferred by the nanoparticle adjuvant was also correlated with IL-6 and IL-10 splenic levels. Each mother was allowed to feed her progeny for one week. Suckling pups presented specific IgA in feces demonstrating their passive immunization through colostrum intake. Two weeks after the pups were born, they were infected orally with a single dose of F4 E. coli (1.2 × 108 CFU/pup). Results showed that 70% of the pups from dams immunized with OMV-NP were protected. In contrast, 80% of the pups from dams immunized with free OMV died as a result of the experimental challenge. These findings support the use of zein nanoparticles coated with a Gantrez-mannosamine shield as adjuvant delivery system for the oral immunization during pregnancy to confer immunity to the offspring through maternal immunization
- Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens(EDP Sciences, 2004) Ochoa-Repáraz, J. (Javier); Sesma, B. (Begoña); Gamazo, C. (Carlos); Alvarez, M. (Miguel); Renedo, M.J. (María Jesús); Irache, J.M. (Juan Manuel)A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.
- Intracellular killing of Brucella melitensis in human macrophages with microspheres-encapsulated gentamicin(Oxford University Press, 2006-06-26) Lecaroz, M.C. (María Concepción); Blanco-Prieto, M.J. (María José); Gamazo, C. (Carlos); Burrell, M.A. (María Ángela)Objectives: Treatment of human brucellosis demands antibiotic targeting into the mononuclearphagocytic system. The aim of this work was to prepare and characterize particulate carriers containing gentamicin and to study their interactions with phagocytic cells and bactericidal activity against intracellular Brucella melitensis. Methods: Different poly(lactide-co-glycolide) (PLGA)polymers with free carboxylic end-group wereusedto formulate micro- and nanoparticles containing gentamicin, by a water-oil-water solvent-evaporation technique. PLGA 502H and 75:25H microparticles were selected because they showed the highest gentamicin loadings as well as good physico-chemical properties and sustained release in vitro. Results: Gentamicin-containing microspheres of both polymers were successfully phagocytosed by infected THP-1 human monocytes, and immunocytochemistry studies revealed that the antibiotic reached Brucella-specific compartments. A dose of 30 mg of encapsulated gentamicin was able to reduce intracellular Brucella infection by 2.2 log. Conclusions: Altogether, these results suggest that 502H and 75:25H microspheres are suitable carriers for gentamicin targeting inside human macrophages and thus for brucellosis treatment.
- Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators(Elsevier, 2011) Mansilla, C. (Cristina); Camacho, A.I. (A.I.); Souza, J. (Juliana) de; Gamazo, C. (Carlos); Tamayo, I. (Ibai); Esparza, I. (Irene); Irache, J.M. (Juan Manuel); Raquel; Lasarte, J.J. (Juan José)Adjuvant research is being oriented to TLR-agonists, but complement activation has been relatively unexplored. In previous studies it was demonstrated that poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (PVMA NPs) used as adjuvant differentially activate dendritic cells through toll like receptors (TLR) stimulation, however, a high dose of these NPs was used. Now, we demonstrated a dose-response effect, with a concentration as low as 20μg/mL able to stimulate TLR2 and TLR4 transfected dendritic cells. In addition, we investigated whether PVMA NPs are able to exploit also the immunomodulatory benefits of complement activation. Results indicated that the hydroxylated surface of these NPs highly activated the complement cascade, as measured by adsorption studies and a complement fixation bioassay. Stable binding of C3b to NPs was confirmed as indicated by lability to SDS treatment after washing resistance. Complement consumption was confirmed as the lytic capacity of complement exposed to NPs was abolished against antibody-sensitized sheep erythrocytes, with a minimal inhibitory concentration of 50μg NPs, equivalent to a surface of 1cm(2). On the contrary, nanoparticles prepared with poly(lactic-co-glycolic acid) (PLGA), used as a reference, did not consume complement at a concentration ≥3mg NPs (≥40cm(2)). Complement consumption was inhibited when PVMA NPs were cross-linked with diamino groups (1,3-diaminopropane), indicating the role of hydroxyl groups as responsible of the phenomenon. These results favour a model whereby PVMA NPs adjuvant activate complement on site to attract immature antigen presenting cells that are activated through TLR2 and TLR4.
- Polymeric carriers for amphotericin B: in vitro activity, toxicity and therapeutic efficacy against systemic candidiasis in neutropenic mice(Oxford University Press, 2003) Campanero, M.A. (Miguel Angel); Espuelas, S. (Socorro); Gamazo, C. (Carlos); Barratt, G. (G.); Legrand, P. (P.); Cheron, M. (M.); Irache, J.M. (Juan Manuel); Appel, M. (M.)Objective: To study the toxicity and activity of two new amphotericin B formulations: poly(ε-caprolactone) nanospheres coated with poloxamer 188 (AmB-NP) and mixed micelles with the same surfactant (AmB-MM). Materials and methods: The toxicity of these formulations was evaluated in erythrocytes, J774.2 macrophages and LLCPK1 renal cells, as well as in mice. Activity was determined in clinical isolates and in neutropenic mice. Mice were made neutropenic with 5-fluorouracil, infected with Candida albicans and treated with the antifungal formulations for three consecutive days. AmB association in cells and accumulation in kidneys and liver of animals was quantified by HPLC. Results: Both formulations decreased between 8- and 10-fold the MIC of the polyene against clinical isolates of C. albicans. However, their activity was lower than or equal to that of AmB-deoxycholate when it was assessed against C. albicans-infected macrophages. When given as a single intravenous dose in mice, AmB-MM and AmB-NP had an LD50 of 9.8 and 18.6 mg/kg, respectively, compared with 4 mg/kg for AmBdeoxycholate. Comparison of residual infection burdens in the liver and kidneys showed that AmB-deoxycholate (0.5 mg/kg) was more effective and faster in eradicating yeast cells than polymeric formulations. This fact can be related to a lower AmB accumulation inside macrophages and in liver and kidneys (about 1.5 mg drug/g tissue) of mice, compared with those detected for AmB-deoxycholate (4 mg drug/g). Overall, the efficacy of these formulations at 2 mg/kg was equal to that of AmB-deoxycholate at 0.5 mg/kg. Conclusions: AmB-MM and AmB-NP decreased the in vivo antifungal activity of AmB, and higher concentrations were therefore necessary to obtain a similar therapeutic effect. However, these higher concentrations were achievable owing to the reduced toxicity of these formulations.
- Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation(American Society of Microbiology, 2010) Ochoa-Repáraz, J. (Javier); Mansilla, C. (Cristina); Gamazo, C. (Carlos); Tamayo, I. (Ibai); Irache, J.M. (Juan Manuel); Lasarte, J.J. (Juan José)The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN- ], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8 T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation.
- Enzyme-linked immunosorbent assay with a Salmonella enteritidis antigen for differentiating infected from vaccinated poultry(BioMed Central, 2000) Sesma, B. (Begoña); Gamazo, C. (Carlos); Alvarez, M. (Miguel); Galindo, J. (Julia); Solano, C. (Cristina); Solsona, M.J. (María J.)The specificity and sensitivity of indirect ELISA, based on the use of four different antigenic extracts obtained from a clinical isolate of Salmonella enteritidis, were compared with those obtained with the gm-flagellin based ELISA (IDEXX). A total of 116 serum samples from salmonellae free, naturally infected and vaccinated hens were studied. The results showed that the indirect ELISA, based on lipopolysaccharide (LPS), O-polysaccharide (PS) or membrane sediment (SD) antigens, enable the identification of a greater number of infected birds and discriminated field antibody responses from vaccinal ones better than the commercial IDEXX test. The indirect ELISA that used a O-polysaccharide rich fraction (PS) proved to be the most specific and sensitive test, suggesting that this indirect ELISA could be used to confirm IDEXX results, especially when the differentiation between vaccinated and infected poultry is required.
- Protective immunity to Brucella ovis in BALB/c mice following recovery from primary infection or immunization with subcellular vaccines(American Society for Microbiology, 1994) Elzer, P.H. (P. H.); Marin, C.M. (C. M.); Gamazo, C. (Carlos); Winter, A.J. (A. J.); Jimenez-de-Bagues, M. (M.); Blasco, J.M. (J. M.)Experiments were performed with BALB/c mice to elucidate the roles of humoral and cell-mediated immune responses in the acquisition of protective immunity to Brucella ovis and to compare infection immunity with immunity developed through vaccination with a hot saline extract (HS) of B. ovis. Mice convalescing from a primary infection with B. ovis displayed a high level of resistance to reinfection, as evidenced by splenic bacterial counts decreased over 10,000-fold from control groups at 2 weeks after challenge. Passive transfer assays revealed that protection was mediated by both T lymphocytes and antibodies but that antibodies had a substantially greater role on the basis of log units of protection that were transferred. Antibodies specific for HS proteins in sera from convalescent mice were predominantly of the immunoglobulin G 2a and 3 isotypes. Vaccination with HS conferred good protection against B. ovis, but protection was greatly enhanced by the incorporation of QS-21 or other adjuvants. Protection provided by the HS vaccine resulted largely from immune responses to its protein moieties. A critical evaluation of the protective efficacy of the rough lipopolysaccharide component of HS was precluded by its poor immunogenicity in BALB/c mice. HS-QS-21 afforded protection against challenge infection with B. ovis as good as that which developed after a primary infection and as good as or better than that provided by attenuated Brucella melitensis vaccine strain Rev 1. Passive transfer experiments confirmed that the magnitudes of both humoral and cell-mediated forms of protective immunity were equivalent in mice vaccinated with HS-QS-21 and those recovering from a primary infection. Protective immunity to B. ovis in mice therefore resembled that to Brucella abortus, except that the relative roles of humoral and cell-mediated immunity, rather than being equivalent, were shifted toward a greater role for antibodies.