RESUMEN
The effect of storage temperature and time on the viability of several foodborne bacterial pathogens inoculated into vacuum-packed canned pasteurized cow's milk cheese was investigated. Three popular cheese styles namely, a semi-soft white Monterey Jack style cheese, and two Cheddar cheeses vacuum packaged in mason jars were inoculated with a 3-strain cocktail of each of the following microbes at the mean concentrations listed: Escherichia coli O157:H7 (6.6â¯logâ¯CFU/g), Salmonella spp. (6.3â¯logâ¯CFU/g), Listeria innocua (6.4â¯logâ¯CFU/g), Staphylococcus aureus (3.6â¯logâ¯CFU/g), and Clostridium sporogenes vegetative cells (6.3â¯logâ¯CFU/g), and spores (6.0â¯logâ¯CFU/g). The effect of storage temperature (at 4.4, 10, and 21.1⯰C) and the time (from 0 to 365â¯days) on the survival of the inoculated organisms was evaluated at different sampling times (0, 30, 60, 120,180, and 365â¯days). Both storage temperature and the time had a significant effect on the viability of the test organisms. Increasing the storage temperature from 4.4 to 21.1⯰C and the storage time for up to 365â¯days increased pathogen reduction. The type of cheese also had a significant effect on the viability of the test organisms. At the same sampling times, the viability of E. coli O157:H7 and Salmonella spp., were highest in Monterey Jack-style cheese followed by the Cheddar cheeses one to which annatto had been added (Cheddar 1) and the second, a white Cheddar that has an added adjunct flavor culture (Cheddar 2). Similarly, the type of cheese and the time-temperature conditions to which the cheese was exposed had a significant effect on the viability of L. innocua. Among the tested organisms, S. aureus was most susceptible while C. sporogenes (both vegetative cells and spores) were most resistant. The findings of this challenge study indicate that vacuum packed canned cheese is not a favorable environment for the growth of bacterial pathogens. Depending upon the type of canned cheese, appropriate storage times and temperatures are critical to ensure microbiological safety.
Asunto(s)
Queso/microbiología , Clostridium/crecimiento & desarrollo , Escherichia coli O157/crecimiento & desarrollo , Listeria monocytogenes/crecimiento & desarrollo , Listeria/crecimiento & desarrollo , Salmonella/crecimiento & desarrollo , Esporas Bacterianas/crecimiento & desarrollo , Staphylococcus aureus/crecimiento & desarrollo , Animales , Bovinos , Recuento de Colonia Microbiana , Femenino , Microbiología de Alimentos , Almacenamiento de Alimentos , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/prevención & control , Leche/microbiología , Temperatura , VacioRESUMEN
Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-γ, TNF-α and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Anticuerpos Antibacterianos/biosíntesis , GMP Cíclico/análogos & derivados , Inmunoglobulina G/biosíntesis , Compuestos de Alumbre/administración & dosificación , Animales , Anticuerpos Antibacterianos/inmunología , GMP Cíclico/administración & dosificación , GMP Cíclico/inmunología , Femenino , Centro Germinal/inmunología , Antígenos de Superficie de la Hepatitis B/administración & dosificación , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunización , Inmunoglobulina G/inmunología , Memoria Inmunológica , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-10/biosíntesis , Interleucina-10/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/inmunología , Macaca mulatta , Ratones , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/inmunología , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Pichia pastoris is a methylotropic yeast that has gained great importance as an organism for protein expression in recent years. Here, we report the expression of recombinant human erythropoietin (rhEPO) in glycoengineered P. pastoris. We show that glycosylation fidelity is maintained in fermentation volumes spanning six orders of magnitude and that the protein can be purified to high homogeneity. In order to increase the half-life of rhEPO, the purified protein was coupled to polyethylene glycol (PEG) and then compared to the currently marketed erythropoiesis stimulating agent, Aranesp(®) (darbepoetin). In in vitro cell proliferation assays the PEGylated protein was slightly, and the non-PEGylated protein was significantly more active than comparator. Pharmacodynamics as well as pharmacokinetic activity of PEGylated rhEPO in animals was comparable to that of Aranesp(®). Taken together, our results show that glycoengineered P. pastoris is a suitable production host for rhEPO, yielding an active biologic that is comparable to those produced in current mammalian host systems.
Asunto(s)
Eritropoyetina/biosíntesis , Pichia/metabolismo , Ingeniería de Proteínas/métodos , Animales , Proliferación Celular/efectos de los fármacos , Darbepoetina alfa , Eritropoyetina/análogos & derivados , Eritropoyetina/sangre , Eritropoyetina/genética , Eritropoyetina/farmacocinética , Eritropoyetina/farmacología , Femenino , Glicosilación , Humanos , Masculino , Ratones , Pichia/genética , Polietilenglicoles , Polisacáridos/química , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
One mechanism to control immune responses following infection is to rapidly down-regulate Ag presentation, which has been observed in acute viral and bacterial infections. In this study, we describe experiments designed to address whether Ag presentation is decreased after an initial response to Leishmania major. Naive alphabeta-Leishmania-specific (ABLE) TCR transgenic T cells were adoptively transferred into mice at various times after L. major infection to determine the duration of presentation of parasite-derived Ags. ABLE T cells responded vigorously at the initiation of infection, but the ability to prime these cells quickly diminished, independent of IL-10, regulatory T cells, or Ag load. However, Ag-experienced clonal and polyclonal T cell populations could respond, indicating that the diminution in naive ABLE cell responses was not due to lack of Ag presentation. Because naive T cell priming could be restored by removal of the endogenous T cell population, or adoptive transfer of Ag-pulsed dendritic cells, it appears that T cells that have previously encountered Ag during infection compete with naive Ag-specific T cells. These results suggest that during L. major infection Ag-experienced T cells, rather than naive T cells, may be primarily responsible for sustaining the immune response.
Asunto(s)
Presentación de Antígeno , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Línea Celular , Proliferación Celular , Regulación hacia Abajo/inmunología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fase de Descanso del Ciclo Celular/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/trasplanteRESUMEN
The importance of the site of Ag localization within microbial pathogens for the effective generation of CD8+ T cells has been studied extensively, generally supporting the view that Ag secretion within infected target cells is required for optimal MHC class I-restricted Ag presentation. In contrast, relatively little is known about the importance of pathogen Ag localization for the activation of MHC class II-restricted CD4+ T cells, despite their clear importance for host protection. We have used the N-terminal targeting sequence of Leishmania major hydrophilic acylated surface protein B to generate stable transgenic lines expressing physiologically relevant levels of full-length OVA on the surface of metacyclic promastigotes and amastigotes. In addition, we have mutated the hydrophilic acylated surface protein B N-terminal acylation sequence to generate control transgenic lines in which OVA expression is restricted to the parasite cytosol. In vitro, splenic dendritic cells are able to present membrane-localized, but not cytosolic, OVA to OVA-specific DO.11 T cells. Strikingly and unexpectedly, surface localization of OVA is also a strict requirement for recognition by OVA-specific T cells (DO.11 and OT-II) and for the development of OVA-specific Ab responses in vivo. However, recognition of cytosolic OVA could be observed with increasing doses of infection. These data suggest that, even under in vivo conditions, where varied pathways of Ag processing are likely to operate, the site of Leishmania Ag localization is an important determinant of immunogenicity and hence an important factor when considering the likely candidacy of vaccine Ags for inducing CD4+ T cell-dependent immunity.
Asunto(s)
Leishmania major/genética , Leishmania major/inmunología , Ovalbúmina/genética , Ovalbúmina/inmunología , Animales , Animales Modificados Genéticamente , Presentación de Antígeno , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , ADN Protozoario/genética , Expresión Génica , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Fracciones Subcelulares/inmunologíaRESUMEN
For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in approximately 85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Pulmón/inmunología , Virus de la Parainfluenza 5/inmunología , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/virología , Infecciones por Rubulavirus/inmunología , Animales , Antígenos Virales/inmunología , Modelos Animales de Enfermedad , Epítopos/inmunología , Femenino , Interferón gamma/biosíntesis , Pulmón/patología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas/inmunología , Infecciones del Sistema Respiratorio/patología , Infecciones por Rubulavirus/patología , Infecciones por Rubulavirus/virología , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/inmunologíaRESUMEN
Alicyclobacillus acidoterrestris, a thermoacidophilic, spore-forming bacterium, has been identified as a spoilage organism in commercial, pasteurized fruit juices. This study was undertaken to evaluate chlorine dioxide for reducing numbers of A. acidoterrestris spores on laboratory media and on apples. A. acidoterrestris spores in aqueous suspension and on apple surfaces of four different cultivars were treated with several concentrations of chlorine dioxide. Spores in aqueous suspension treated with 40 ppm for 5 min were reduced by more than 4 log. Treatment with 80 ppm for 1 min and 120 ppm for 30 s resulted in about 1.8 log and 4.8 log reductions of spore viability, respectively, and treatment at 80 and 120 ppm for 5 min reduced spore viability to undetectable levels (<0.7 log CFU/ml). When applied to the surfaces of four different apple cultivars ('Red Delicious', 'Golden Delicious', 'Gala', and 'Fuji'), 40 ppm free chlorine dioxide reduced A. acidoterrestris spore numbers by 1.5, 3.2, 4.5, >4.8 log after 1-, 2-, 3-, and 4-min treatments, respectively. Spore numbers were reduced by >4.8 log with 120 ppm free chlorine dioxide after only 1-min treatment. However, there was no significant difference between apple cultivars (P>0.05) on spore reduction. These results show the great effectiveness of chlorine dioxide in controlling A. acidoterrestris spores both in aqueous suspension and on apple surfaces. There was no synergistic effect on spore reduction when chlorine dioxide treatment of aqueous suspension was followed by heat.
Asunto(s)
Compuestos de Cloro/farmacología , Microbiología de Alimentos , Bacilos Grampositivos Formadores de Endosporas/crecimiento & desarrollo , Calor , Malus/microbiología , Óxidos/farmacología , Bebidas/microbiología , Recuento de Colonia Microbiana , Relación Dosis-Respuesta a Droga , Conservación de Alimentos/métodos , Bacilos Grampositivos Formadores de Endosporas/efectos de los fármacos , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/crecimiento & desarrollo , Factores de TiempoRESUMEN
The increased potency of high avidity CD8+ T cells for the clearance of viral infections has been well documented. We have previously reported the novel finding that intranasal infection with the paramyxovirus SV5 induces a CD8+ T cell response to the SV5 P protein that is almost exclusively of high avidity. Based on our results that the level of peptide presentation is a critical factor in the selective expansion of high versus low avidity cells in vitro, we hypothesized that the avidity of the anti-viral response generated in vivo could be altered by increasing the turnover of the P protein during viral infection through linkage to ubiquitin (UbP). Infection with a virus expressing UbP (VV-UbP) elicited a significant increase in low avidity cells in both BALB/c and C3H mice compared to the almost exclusively high avidity response elicited by VV-P. Our results are the first demonstration of the control of avidity during the antiviral response through an engineered change to a viral antigen. The implications of our findings for vaccine development are discussed.
Asunto(s)
Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Virus de la Parainfluenza 5/inmunología , Infecciones por Rubulavirus/inmunología , Infecciones por Rubulavirus/virología , Animales , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Semivida , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Virus de la Parainfluenza 5/metabolismo , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Unión Proteica , Proteínas de Unión al ARN , Infecciones por Rubulavirus/metabolismo , Ubiquitina/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
A new rapid method for monitoring coliforms was developed on the basis of the instant gelling effects of alginate and calcium. The effectiveness of this new method in the detection of coliforms was evaluated. Tests involving Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, total coliforms in milk, cold-injured coliforms, and total coliforms in raw milk were carried out. The bacterial samples were diluted in 0.2% peptone water containing 90 mM CaCl2 and added into test tubes containing modified purple broth base medium. Coliform concentrations were determined on the basis of the time of color change and gas production in the alginate tubes. All results obtained by the alginate method correlated strongly with those obtained by the conventional violet red bile agar (VRBA) plating method. The alginate method reduced detection time by 12 to 14 h compared with the conventional VRBA plating method. The alginate method can be applied in field studies more easily than melted-agar systems can. The results of this study indicate that the alginate method is an accurate, rapid, simple, and economical way to monitor and estimate concentrations of total coliforms in food.
Asunto(s)
Alginatos , Recuento de Colonia Microbiana/métodos , Enterobacteriaceae/aislamiento & purificación , Ácido Glucurónico , Ácidos Hexurónicos , Leche/microbiología , Animales , Técnicas Bacteriológicas , Calcio/metabolismo , Enterobacteriaceae/metabolismo , Fermentación , Microbiología de Alimentos , Geles , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Following intranasal administration, the model paramyxovirus simian virus 5 (SV5) establishes an infection in the respiratory tract of mice, which is subsequently cleared by CD8+ T cells. In this study, we sought to understand the maturation of the antiviral immune response over time by assessing the functional avidity of the responding T cells and the expansion of immunodominant populations. Surprisingly, we determined that the initial response to Ag at day 3 (d3) in the mediastinal lymph node was exclusively high avidity. However, by d5 postinfection, low avidity cells were approximately 50% of the responding T cell population. Following secondary exposure to SV5, high avidity CD8+ T cells again are the exclusive cell type present at early times postinfection (d2). Similarly, high avidity cells were preferentially elicited at d3 following infection with the unrelated vaccinia virus. We also made the observation that the immunodominance profile has not been established at d3 postinfection with SV5. However, by d5 a clear immunodominance pattern arises and is permanently maintained. These data indicate that high avidity cells are the predominant population responding at early times postinfection following respiratory infection with SV5 or vaccinia virus. However, as the response progresses, low avidity cells are activated/expanded to a greater extent compared with high avidity cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Activación de Linfocitos , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/virología , Infecciones por Respirovirus/inmunología , Subgrupos de Linfocitos T/inmunología , Administración Intranasal , Animales , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Células Cultivadas , Inmunización , Inmunización Secundaria , Epítopos Inmunodominantes/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Mediastino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Respirovirus/inmunología , Infecciones por Respirovirus/virología , Subgrupos de Linfocitos T/virología , Células Tumorales Cultivadas , Vaccinia/inmunología , Vaccinia/virología , Proteínas Virales/inmunologíaRESUMEN
Developments in our ability to integrate and analyze data held in existing heterogeneous data resources can lead to an increase in our understanding of biological function at all levels. However, supporting ad hoc queries across multiple data resources and correlating data retrieved from these is still difficult. To address this, we are building a mediator based on the functional data model database, P/FDM, which integrates access to heterogeneous distributed biological databases. Our architecture makes use of the existing search capabilities and indexes of the underlying databases, without infringing on their autonomy. Central to our design philosophy is the use of schemas. We have adopted a federated architecture with a five-level schema, arising from the use of the ANSI-SPARC three-level schema to describe both the existing autonomous data resources and the mediator itself. We describe the use of mapping functions and list comprehensions in query splitting, producing execution plans, code generation, and result fusion. We give an example of cross-database querying involving data held locally in P/FDM systems and external data in SRS.
Asunto(s)
Algoritmos , Sistemas de Administración de Bases de Datos , Técnicas de Apoyo para la Decisión , Almacenamiento y Recuperación de la Información/métodos , Inteligencia Artificial , Biología Computacional , Redes de Comunicación de Computadores , Bases de Datos Factuales/clasificación , Estudios de Factibilidad , InternetRESUMEN
A new rapid coliform-monitoring method using thin tube cultivation (TTC) was developed. One hundred microliters of modified selective medium (1.5% agar) and 100 microl of sample were mixed in thin cultivation tubes, yielding a semisolid mixture of 0.75% agar. The tubes were incubated at 37 degrees C and checked for a positive reaction (gas production from lactose fermentation and color changes from acid production) at 30-min intervals. The positive reaction times for TTC were compared with actual coliform numbers. The first experiment was performed to validate the TTC method using pure coliform cultures (the Escherichia coli group, the Enterobacter cloacae group. and the Klebsiella pneumoniae group). The results indicated that the TTC method saved about 10 to 14 h of incubation time compared with the conventional plating method. When coliforms were present in small numbers, such as I to 1.5 log10 CFU/ml, the TTC method detected their presence within 10 h. When highly contaminated samples were assayed (6 log10 CFU/ml), the TTC method detected the presence of coliforms within 4 to 5 h. In a food system experiment, pasteurized skim milk inoculated with a coliform mixture was assayed by the TTC method. The coliforms in the milk were quickly detected, resulting in a savings of 11 to 14 h compared with the conventional plating method. Cold-injured coliforms were studied to evaluate the TTC system. The recovery and estimation of injured coliforms by the TTC method strongly agree with those of the conventional plating method. The TTC system was also applied to ascertain the levels of natural coliforms in noninoculated milk. The TTC system reduced incubation times by about 11 to 14 h compared with the conventional plating method. According to these data, this method is a rapid, simple, accurate, and economical way to monitor and estimate total coliforms in milk.