RESUMEN
BACKGROUND: In the absence of breast-feeding and its immunomodulatory factors, supplementation of starter infant formula (IF) with probiotics is currently used to support immune functions and gut development. AIM: To assess whether immune-related beneficial effects of regular dose (10(7) CFU/g of powder) of the probiotic Bifidobacterium lactis CNCM I-3446 (hereafter named B. lactis) in starter IF supplementation can be maintained with starter IF containing a low dose (10(4) CFU/g of powder) of B. lactis. METHOD: This trial was designed as a pilot, prospective, double-blind, randomized, single-center clinical trial of two parallel groups (n = 77 infants/group) of C-section delivered infants receiving a starter IF containing either low dose or regular dose of the probiotic B. lactis from birth to six months of age. In addition, a reference group of infants breast-fed for a minimum of four months (n = 44 infants), also born by C-section, were included. All groups were then provided follow-up formula without B. lactis up to 12 months of age. Occurrence of diarrhea, immune and gut maturation, responses to vaccinations, and growth were assessed from birth to 12 months. The effect of low-dose B. lactis formula was compared to regular-dose B. lactis formula, considered as reference for IF with probiotics, and both were further compared to breast-feeding as a physiological reference. RESULTS: Data showed that feeding low-dose B. lactis IF provides similar effects as feeding regular-dose B. lactis IF or breast milk. No consistent statistical differences regarding early life protection against gastrointestinal infections, immune and gut maturation, microbiota establishment, and growth were observed between randomized formula-fed groups as well as with the breast-fed reference group. CONCLUSION: This pilot study suggests that supplementing C-section born neonates with low-dose B. lactis-containing starter formula may impact immune as well as gut maturation similarly to regular-dose B. lactis, close to the breast-feeding reference.
RESUMEN
The goal of the present paper was to optimize the encapsulation of beta-lactoglobulin (BLG) within poly(lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion solvent evaporation method. The role of the pH of the external phase and the introduction of the surfactant Tween 20, in the modulation of the entrapment and release of BLG from microparticles, was studied. Reducing the solubility of BLG by decreasing the pH of the external phase to a value close to the pI of BLG resulted in a better encapsulation with, however, a larger burst release effect. By contrast, Tween 20 was shown to increase the encapsulation efficiency of BLG and reduce considerably the burst release effect. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface. In addition, Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communicating with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease in the burst release. These results constitute a step ahead in the improvement of an existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.
Asunto(s)
Ácido Láctico/administración & dosificación , Lactoglobulinas/administración & dosificación , Ácido Poliglicólico/administración & dosificación , Polímeros/administración & dosificación , Concentración de Iones de Hidrógeno , Lactoglobulinas/química , Microesferas , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
Allergies to milk proteins are frequently encountered in the new born population. In order to prevent this allergy by inducing oral tolerance, one of the major allergenic milk protein, B lactoglobulin (BLG) was entrapped into biodegradable Poly (lactide-co-glycolide) microspheres and was then orally given to mice. Microspheres are able to protect proteins against degradation by intestinal proteolytic enzymes and to target the Peyers patches which are one important priming site of the mucosal immune system. Microspheres were prepared by the multiple emulsion solvent evaporation method. The goal of the formulation study was to associate large amounts of proteins to the smallest amount of polymer so that a minimal quantity of microspheres would be administered. It was shown that introducing tween 20 in the formulation was able to increase the encapsulation efficiency and to better control protein release reducing the burst release effect. Moreover, Oral administration of microspheres containing BLG reduced significantly (by 10.000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. In conclusion, microspheres appear to be optimal systems to induce oral tolerance.
Asunto(s)
Ácido Láctico , Lactoglobulinas/uso terapéutico , Intolerancia a la Lactosa/tratamiento farmacológico , Ácido Poliglicólico , Polímeros , Portadores de Fármacos , Composición de Medicamentos , Excipientes , Humanos , Inmunidad/efectos de los fármacos , Lactoglobulinas/administración & dosificación , Microesferas , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
The recognition that Helicobacter pylori is associated with an array of gastric disorders immediately raises several issues with regard to the role of the local immune system. The belief that the harsh gastric environment limits or prevents infection has perhaps dismissed studies into the immunology of the stomach as a low priority. Now, in combination with our understanding of local immune reactivity in pernicious anaemia, an interest in defining the contribution of the immune response in the pathogenesis of disease associated with H. pylori has fueled a great deal of interest. Furthermore, the evidence of local immunity to this bacteria has kindled interest that gastric immune/inflammatory responses may contribute to the treatment or prevention of a gastric infection.
Asunto(s)
Mucosa Gástrica/inmunología , Helicobacter pylori/inmunología , Mucosa Intestinal/inmunología , Animales , Infecciones por Helicobacter/inmunología , Humanos , InmunidadAsunto(s)
Formación de Anticuerpos , Antígenos CD/inmunología , Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/inmunología , Inmunoterapia Adoptiva , Animales , Antígenos CD5 , Células Cultivadas , Tolerancia Inmunológica , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Depleción Linfocítica , Ratones , Ratones Endogámicos CBA , Ratones MutantesAsunto(s)
Linfocitos B/inmunología , Eritrocitos/inmunología , Tolerancia Inmunológica , Síndromes de Inmunodeficiencia/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/inmunología , Subgrupos de Linfocitos B/inmunología , Antígenos CD5 , Isotipos de Inmunoglobulinas/metabolismo , Inmunoglobulinas/metabolismo , Síndromes de Inmunodeficiencia/genética , Macrófagos/inmunología , Ratones , Ratones Endogámicos CBA , Ratones MutantesRESUMEN
High concentrations of ciprofloxacin have been shown to persist in the faeces of volunteers for several days after a week of oral treatment with this drug, which was also found to have a prolonged effect on aerobic Gram-negative intestinal bacteria. To determine whether a shorter course of ciprofloxacin would have the same prolonged effect, we treated ten healthy adult volunteers with a single oral dose of 750 mg ciprofloxacin and found that this was not followed by any significant changes in the counts of anaerobes or streptococci, but that there was a mean decrease of 2.5 log10 cfu/ml in the counts of faecal Enterobacteriaceae, which lasted for a full week. We attributed this to the persistence of high faecal ciprofloxacin concentrations for several days in all the volunteers. We did not observe any significant increase in the MICs of ciprofloxacin for faecal Enterobacteriaceae, or any faecal overgrowth of staphylococci, fungi, Pseudomonas aeruginosa or Clostridium difficile.
Asunto(s)
Ciprofloxacina/farmacocinética , Heces/microbiología , Adulto , Bacterias Anaerobias/efectos de los fármacos , Ciprofloxacina/sangre , Ciprofloxacina/farmacología , Enterobacteriaceae/efectos de los fármacos , HumanosRESUMEN
Intestinal endogenous members of the family Enterobacteriaceae were eliminated in 12 human volunteers treated with 400 or 800 mg of oral norfloxacin per day for 5 days. No clones resistant to quinolone derivatives were isolated. Counts of aerotolerant streptococci were affected to various degrees, depending on their susceptibility to norfloxacin. During treatment, counts of anaerobes remained above 9.8 log10 CFU/g of feces. A total of 932 anaerobic isolates from the predominant flora (over 10(9) CFU/g) in fecal samples obtained before or during norfloxacin treatment were classified by a simple morphological and physiological scheme. The composition of this flora was fairly stable from one sample to another before treatment and was not substantially modified by norfloxacin. Intestinal resistance to colonization by exogenous microorganisms was studied in gnotobiotic mice associated with a human fecal flora. The composition of the fecal flora of the human donor and the fecal concentrations of norfloxacin in the volunteers were reproduced in the intestine of the mice. Resistance to colonization by exogenous microorganisms was reduced by norfloxacin for only 2 of 14 (14%) of the strains tested. These results suggest that norfloxacin is a good candidate for selective antimicrobial modulation of the intestinal tract in humans.
Asunto(s)
Antiinfecciosos/farmacología , Heces/microbiología , Intestinos/microbiología , Norfloxacino/farmacología , Animales , Vida Libre de Gérmenes , Humanos , Ratones , Ratones Endogámicos C3H , Ácido Nalidíxico/farmacología , Factores de TiempoRESUMEN
Intestinal members of the family Enterobacteriaceae were eliminated in five human volunteers treated with oral ofloxacin for 5 days. No emergence of resistant Enterobacteriaceae was observed. Counts of group D streptococci were significantly reduced. Colonization by Candida sp. was observed in all five volunteers during ofloxacin treatment. The anaerobic flora was fairly stable from one sample to another before treatment and was not substantially modified by ofloxacin.
Asunto(s)
Antiinfecciosos/farmacología , Enterobacteriaceae/efectos de los fármacos , Heces/microbiología , Oxazinas/farmacología , Administración Oral , Antibacterianos , Candida/aislamiento & purificación , Farmacorresistencia Microbiana , Humanos , Ofloxacino , Oxazinas/administración & dosificaciónRESUMEN
The observation that approximately half of the B cells in the murine intestinal lamina propria are derived from peritoneal CD5 B-cell precursors raises the question of their contribution to mucosal protection. Using mice with X-linked immunodeficiency which are deficient in CD5+ B cells, we showed that they mount little serum and virtually no intestinal immunoglobulin M (IgM), IgG, and IgA antibody responses following oral inoculation with live Salmonella typhimurium. Nonresponsive Xid mice were reconstituted with responsive CBA/Ca donor cell preparations which were constitutively enriched or depleted of CD5 B-cell precursors. Reconstitution of irradiated Xid mice with CD5 B-cell-deficient bone marrow from CBA/Ca donors marginally improved IgM responses in the intestinal mucosa but had no effect on IgG or IgA in response to oral immunization with live S. typhimurium. Whenever Xid mice were reconstituted with donor cells from the peritoneal cavity, which are enriched for CD5 B-cell precursors, strong IgA and in some cases IgG responses in the intestinal mucosa were stimulated in response to oral immunization. When mucosal and serum antibody responses were compared, the peritoneal donor cells again reinstated maximal serum antibody responses to S. typhimurium. Serum and mucosal responses to the bacterial hapten phosphorylcholine could be induced in Xid mice after immunization with S. typhimurium or hapten-carrier conjugates but only following reconstitution with donor cells containing CD5 B-cell precursors. These observations suggest that different lymphoid compartments are enriched for regulatory or effector cells which vary in their contributions to the mucosal antibody response against epitopes on S. typhimurium.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Linfocitos B/fisiología , Haptenos/inmunología , Células Madre Hematopoyéticas/fisiología , Síndromes de Inmunodeficiencia/inmunología , Mucosa Intestinal/inmunología , Fosforilcolina/inmunología , Salmonella typhimurium/inmunología , Animales , Formación de Anticuerpos , Ligamiento Genético , Ratones , Ratones Endogámicos CBA , Cavidad Peritoneal/citología , Cromosoma XRESUMEN
When healthy volunteers were given a daily dose of 3 x 10(8) life-dehydrated Saccharomyces cerevisiae cells for 5 days, the volunteers excreted 10(5) living yeast cells per g of feces at first, but the yeast cells disappeared within 5 days of the end of treatment. In gnotobiotic mice, S. cerevisiae administered alone colonized the intestinal tract but did not interfere with previous or subsequent colonization by a variety of potentially enteropathogenic microorganisms. When these microorganisms were present, the intestinal counts of S. cerevisiae were greatly reduced.
Asunto(s)
Intestinos/microbiología , Saccharomyces cerevisiae/metabolismo , Adulto , Animales , Recuento de Colonia Microbiana , Heces/microbiología , Vida Libre de Gérmenes , Humanos , Absorción Intestinal , Intestinos/ultraestructura , Cinética , Ratones , Ratones Endogámicos C3H , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructuraRESUMEN
The ecological impact of roxithromycin given orally at 300 mg/day on the intestinal floras in six human volunteers was studied. The resulting fecal concentrations of active roxithromycin were in the range of 100 to 200 micrograms/g of feces. Consecutive modifications in the composition of the fecal floras were limited to a decrease in counts of total members of the family Enterobacteriaceae. The rest of the intestinal floras, including the predominant anaerobic floras, changed little. No overgrowth of Pseudomonas aeruginosa, staphylococci, fungi, or highly erythromycin-resistant strains of the family Enterobacteriaceae was observed. The strains of Enterobacteriaceae and of anaerobes isolated during treatment were not markedly more resistant to roxithromycin than those isolated before treatment started. Changes in intestinal resistance to colonization by exogenous microorganisms in gnotobiotic mice inoculated with human fecal flora were studied and were also found to be minimal. The impact of oral roxithromycin on the intestinal microbiota appears to be weaker than that previously observed with oral erythromycin, perhaps because the concentrations of roxithromycin in the feces were lower than those previously found for erythromycin.
Asunto(s)
Bacterias/efectos de los fármacos , Heces/microbiología , Intestinos/efectos de los fármacos , Roxitromicina/farmacología , Administración Oral , Adulto , Animales , Bacterias/crecimiento & desarrollo , Recuento de Colonia Microbiana , Vida Libre de Gérmenes , Humanos , Intestinos/microbiología , Ratones , Ratones Endogámicos C3H , Pruebas de Sensibilidad Microbiana , Roxitromicina/sangreRESUMEN
BACKGROUND: Oral administration of an antigen has been shown to suppress the specific immune response to this antigen. This approach, called oral tolerance, has been demonstrated with intact proteins in animal models for prevention of allergy and autoimmune diseases. OBJECTIVE: The purpose of this study was to determine whether oral tolerance can be induced with protein peptides. Partially hydrolyzed and extensively hydrolyzed cow's milk formulas were compared for their capacity to induce tolerance to cow's milk proteins. METHODS: Five-week-old Sprague-Dawley rats were fed cow's milk formulas ad libitum from day 1 to day 19. All animals were immunized with beta-lactoglobulin and ovalbumin on day 5 and bled on day 19. Sera were analyzed for specific IgE and IgG antibodies by ELISA and for functional IgE response by in vitro mast cell mediator (tritiated serotonin) release. In vivo modulation of intestinal mast cells was analyzed by the specific release of the rat mast cell protease II, and T-cell response was determined by tritiated thymidine incorporation into lymph node lymphocytes. RESULTS: Oral administration of a partially hydrolyzed cow's milk formula suppresses specific serum IgE and IgG anti-beta-lactoglobulin antibodies, as well as mediator release from rat mast cells and T-lymphocyte response. This suppression was shown to be antigen-specific and dose-dependent. An extensively hydrolyzed formula was unable to achieve the induction of such an oral tolerance. CONCLUSION: These results support the view that partially hydrolyzed proteins are able to induce specific oral tolerance, whereas extensively hydrolyzed proteins are not.
Asunto(s)
Tolerancia Inmunológica , Lactoglobulinas/inmunología , Proteínas de la Leche/farmacología , Administración Oral , Animales , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina E/sangre , Mucosa Intestinal/citología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Mastocitos/metabolismo , Leche/química , Ratas , Ratas Sprague-Dawley , Proteína de Suero de LecheRESUMEN
BACKGROUND: Oral tolerance against food proteins has been achieved in different animal models with use of native or moderately hydrolyzed proteins as inducers. However, native proteins remain highly allergenic, although it has been demonstrated that protein hydrolyzates and resulting peptides can lose their allergenicity. OBJECTIVE: This study was designed to evaluate the ability of beta-lactoglobulin hydrolyzate and peptides to induce oral tolerance to native beta-lactoglobulin and to identify tolerogenic beta-lactoglobulin peptides with low allergenicity. METHODS: beta-Lactoglobulin was hydrolyzed by trypsin and fractionated by ion exchange chromatography. Peptide enrichment of fractions was evaluated. Balb/c mice were fed beta-lactoglobulin hydrolyzate or fractions by single gavage at day 1. Five days later animals were challenged intraperitoneally with native beta-lactoglobulin. At day 27 delayed-type hypersensitivity was performed. Twenty-four hours later mice were bled, and intestinal contents and spleens were collected. Oral tolerance was measured by titrating specific IgE in sera and intestinal samples. Specific T-cell responses were analyzed by splenocyte proliferation. Antigenicity of hydrolyzate and fractions was evaluated by specific ELISA inhibition. RESULTS: Mice fed either beta-lactoglobulin hydrolyzate or 2 fractions of the hydrolyzate were tolerized against beta-lactoglobulin. Specific serum and intestinal IgE were suppressed. Delayed-type hypersensitivity and proliferative responses were inhibited. One tolerogenic fraction was found to be 50 times less antigenic than the total beta-lactoglobulin hydrolyzate was. CONCLUSION: These findings support the strategy of inducing oral tolerance in "at-risk" patients by means of tolerogenic cow's milk peptides or hydrolyzate.
Asunto(s)
Lactoglobulinas/metabolismo , Biosíntesis de Péptidos/inmunología , Biosíntesis de Péptidos/fisiología , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Antígenos/inmunología , Bovinos , Femenino , Hidrólisis , Tolerancia Inmunológica , Inmunidad Celular/efectos de los fármacos , Inmunización , Ratones , Ratones Endogámicos BALB C , Factores Supresores Inmunológicos/farmacologíaRESUMEN
This study was designed to confirm the tolerogenic properties of beta-lactoglobulin in a mouse model and to assess specific oral tolerance induction in humoral and cellular compartments. BALB/c mice were fed beta-lactoglobulin (BLG) or whey proteins at different ages and subsequently intraperitoneally challenged 5 days later with both BLG and a non-specific antigen, ovalbumin (OVA). Three weeks later, oral tolerance induction was analysed in CMP-fed, versus saline-fed mice, by measuring specific seric and intestinal antibody responses, delayed-type hypersensitivity (DTH), specific splenocyte proliferation, and cytokine secretion patterns. Three-week-old mice fed high doses of either whey proteins or BLG (respectively 3 mg/g or 5 mg/g of body weight) were found to achieve oral tolerization. At humoral and mucosal levels, anti-BLG immunoglobulin E (IgE) were suppressed in these groups when compared with saline fed mice. With respect to cellular responses, systemic DTH and lymphocyte proliferation to BLG were also inhibited in CMP-fed mice. Weaning time was determined to be the best period for oral tolerance induction. Kinetic analyses showed however, that a minimum of 2 weeks was required for oral tolerance detection. Finally, cytokine profiles indicated a reciprocal decrease of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) versus an increase of IL-10 and transforming growth factor-beta (TGF-beta) secretions in tolerized mice. Taken together, these results clearly showed that oral administration of high doses of cows' milk proteins can induce significant hyposensitization in mice, in a specific inhibition of T helper 1 (Th1) lymphocytes with the participation of suppressor cytokines.
Asunto(s)
Alérgenos/administración & dosificación , Citocinas/inmunología , Tolerancia Inmunológica , Inmunoglobulina E/inmunología , Lactoglobulinas/administración & dosificación , Factores de Edad , Alérgenos/inmunología , Análisis de Varianza , Animales , Formación de Anticuerpos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunidad Mucosa , Interferón gamma/análisis , Interleucina-10/análisis , Interleucina-2/análisis , Lactoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas de la Leche/administración & dosificación , Proteínas de la Leche/inmunología , Células TH1/inmunología , Factor de Crecimiento Transformador beta/análisisRESUMEN
The purpose of this study was to investigate the in-vitro interaction between [3H]ampicillin-loaded polyisohexylcyanoacrylate nanoparticles and murine macrophages (peritoneal and J774) infected with Salmonella typhimurium. The multiplicity of infection was ten bacteria to each macrophage and the mean (+/- S.D.) diameter of the nanoparticles was 220 (+/- 20 nm), corresponding to an ampicillin concentration of 2 g/L. The uptake of nanoparticle-bound [3H]ampicillin by non-infected J774 and peritoneal macrophages was six- and 24-fold greater respectively than that of free [3H]ampicillin. For infected cells, uptake by J774 and peritoneal macrophages was nine- and 20-fold greater respectively. However, there was no difference between nanoparticle-bound ampicillin and free ampicillin in terms of bactericidal activity against intracellular S. typhimurium. This unexpected observation might be accounted for by bacterium-induced inhibition of phagosome-lyosome fusion within the macrophages, thereby preventing contact between the bacteria in the phagosomes and the nanoparticles in the secondary lysosomes.
Asunto(s)
Ampicilina/farmacocinética , Macrófagos/metabolismo , Salmonella typhimurium/efectos de los fármacos , Ampicilina/administración & dosificación , Ampicilina/farmacología , Animales , Células Cultivadas , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , RatonesRESUMEN
Oral administration of antigen is known to be appropriate for some vaccine purposes as well as oral tolerance induction. In the present study, oral administration of beta-lactoglobulin (BLG) loaded poly(D,L-lactide-co-glycolide) (D,L-PLG) microspheres induced tolerance was evaluated. A single feeding of 5 micrograms of encapsulated BLG tolerized BALB/c mice to subsequent BLG parenteral challenge, suppressing the specific humoral, intestinal and cellular responses. The tolerogenic efficient dose was then reduced 10,000 times, compared to oral administration of soluble BLG. This suggests that loading food proteins into D,L-PLG microspheres might be a potential tool for inducing oral tolerance with allergens.
Asunto(s)
Materiales Biocompatibles/administración & dosificación , Tolerancia Inmunológica , Ácido Láctico/administración & dosificación , Lactoglobulinas/administración & dosificación , Lactoglobulinas/inmunología , Ácido Poliglicólico/administración & dosificación , Polímeros/administración & dosificación , Animales , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Femenino , Hipersensibilidad Tardía/inmunología , Inmunoglobulina E/biosíntesis , Intubación Gastrointestinal , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Microesferas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Bazo/citología , Bazo/inmunologíaRESUMEN
The milk model protein, beta lactoglobulin (BLG), was encapsulated into microspheres prepared by a multiple emulsion/solvent evaporation method. The effect of the pH of the outer aqueous phase on protein encapsulation and release as well as on microsphere morphology has been investigated. At all tested pH values, the encapsulation efficiency was shown to decrease with increasing the initial amount of BLG. This was correlated with the reduced stability of the primary emulsion as the initial BLG increased. In addition, reducing the solubility of BLG in the external aqueous phase by decreasing the pH to the isoelectric point of BLG (pI 5.2) resulted in an improved protein encapsulation. Moreover, it was shown that combining pH modification and optimal stability of the first emulsion yielded microspheres with a high encapsulation efficiency. However, release kinetic studies revealed that a significant burst release was observed with microspheres loaded with large amounts of BLG, especially when prepared in a medium at pH 5.2. This burst effect was attributed to morphology changes in the microsphere surface which was characterized by the presence of pores or channels able to accelerate the release of BLG. These pores were assumed to result from the presence of large amounts of protein molecules on the microsphere surface, that aggregate during microsphere formation at pH 5.2. Indeed, single adsorption experiments have shown that BLG had a higher affinity for the particle surface when the pH was close to the pI. Thus, reducing the solubility of a protein in the external aqueous phase allows the product of microspheres with a better encapsulation efficiency, although this benefit is provided by a strong adsorption of the protein on microsphere surface.
Asunto(s)
Materiales Biocompatibles/química , Ácido Láctico/química , Ácido Poliglicólico/química , Polímeros/química , Proteínas/química , Adsorción , Tampones (Química) , Emulsiones , Concentración de Iones de Hidrógeno , Cinética , Lactoglobulinas/química , Microscopía Electrónica , Microesferas , Tamaño de la Partícula , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , SolventesRESUMEN
In order to determine the best strategy for detection of aerobactin in members of the family Enterobacteriaceae, we compared the results of three phenotypic assays, including a chemical assay, a cross-feeding bioassay, and an enzyme-linked immunosorbent assay (ELISA), with the results of a dot blot hybridization assay using a specific probe for the aerobactin genes. The sensitivity and specificity of the ELISA were better than those of the chemical and cross-feeding assays, but the results of dot blot hybridization were the most reproducible. However, none of the Serratia and Enterobacter cloacae strains which produced aerobactin hybridized with the probe. We concluded that the best strategy for aerobactin detection is a two-step procedure that combines screening by dot blot hybridization with an ELISA for negative strains.
Asunto(s)
Enterobacteriaceae/química , Ácidos Hidroxámicos/análisis , Sideróforos/análisis , Bioensayo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Ensayo de Inmunoadsorción Enzimática , Ácidos Hidroxámicos/metabolismo , Immunoblotting , Hibridación de Ácido Nucleico , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Sideróforos/genética , Sideróforos/metabolismoRESUMEN
Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.