RESUMEN
BACKGROUND: Sensitization to food allergens indicates the production of food-specific IgE; however, sensitization is not a definite indicator of allergic reaction upon ingestion (N Engl J Med, 344, 2001, 30: J Allergy Clin Immunol, 120, 2007, 491). Currently, food challenge is the best approach to identify the presence or absence of allergy. While 95% positive predictive values (PPVs) thresholds for sIgE can assist with identifying increased likelihood of allergy among those who are sensitized, there are no specific biological markers that differentiate between allergic and sensitized individuals. OBJECTIVES: To determine whether plasma serum cytokine profiles predict (i) sensitization to peanut and egg and (ii) food allergy among sensitized infants. METHODS: Peanut-sensitized (PT) and egg-sensitized 14-month-old infants and nonsensitized controls enrolled in HealthNuts, a population-based study of food allergy, underwent an oral food challenge (OFC). Blood was collected within 1 h after OFC. Serum levels of Th1, Th2 and regulatory cytokines were determined in allergic (n = 79), sensitized (n = 40) and nonsensitized, nonallergic (n = 37) infants by multiplex assay. RESULTS: Food-sensitized infants had significantly higher plasma IL-4, IL-13, IL-12p70 and lower IL-10 levels compared to nonsensitized infants. IL-10 and IL-6 levels were significantly higher in sensitized compared with allergic infants. Egg-allergic infants had significantly higher IL-13 and IL-12p70 levels compared to peanut-allergic (PA) infants. CONCLUSION: Levels of Th2-related cytokines in plasma are higher in food-sensitized infants, irrespective of clinical food allergy status. In contrast, IL-10 levels appear to predict food allergy among sensitized infants. Differences in IL-13 and IL-12p70 between egg- and peanut-allergic infants could help explain the different resolution rates of the allergies.
Asunto(s)
Citocinas/sangre , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/inmunología , Alérgenos/efectos adversos , Alérgenos/inmunología , Arachis/efectos adversos , Estudios de Cohortes , Hipersensibilidad al Huevo/sangre , Hipersensibilidad al Huevo/epidemiología , Hipersensibilidad al Huevo/inmunología , Huevos/efectos adversos , Hipersensibilidad a los Alimentos/epidemiología , Humanos , Lactante , Hipersensibilidad al Cacahuete/sangre , Hipersensibilidad al Cacahuete/epidemiología , Hipersensibilidad al Cacahuete/inmunología , Vigilancia de la PoblaciónRESUMEN
A novel rapid green one-step method is developed for the preparation of bio-silica coated with amorphous MnO2 nanoparticles by treating bio-silica with an acidic permanganate solution. The method developed has the advantage of selectively coating the surface of either one or both sides of the porous silica structure with a thin catalytic active amorphous MnO2 layer in a controlled way. The uncoated and MnO2 coated bio-silica are characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The catalytic activity of amorphous MnO2-coated bio-silica is examined by degrading organic dye at ambient condition. The as-synthesized samples show highly efficient and rapid degradation of Rhodamine B. The simplicity and cost-effectiveness of the materials and method can be very useful for highly efficient degradation of organic pollutants for environmental remediation.
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Contaminantes Ambientales/química , Compuestos de Manganeso/química , Compuestos Orgánicos/química , Óxidos/química , Dióxido de Silicio/química , Catálisis , Restauración y Remediación Ambiental/métodos , Nanopartículas del Metal , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Difracción de Rayos XRESUMEN
Monosized sodium alginate microdroplets are prepared using a flow-focusing microdevice by adjusting the flow rate of the continuous phase (soybean oil) and the dispersed phase (sodium alginate solution). The gelation process of the semi-product, sodium alginate microdroplets, occurs outside the channel in a calcium chloride solution to form tadpole-shaped calcium alginate microparticles. The microparticles prepared are in the range of 100-250 µm in diameter, depending on the experimental conditions. The shape, size and size distribution of these calcium alginate microparticles depend strongly on the calcium solution concentration and the stirring mode. The shaping mechanism of the microparticles and the impact of the experimental conditions on particle shape and size are investigated.
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Alginatos/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Técnicas Analíticas MicrofluídicasRESUMEN
It is generally known that intracellular pH (pH(i)) plays a vital role in cell physiology and that pH(i) homeostasis is essential for normal cellular functions. Therefore, it is desirable to know the pH(i) during cell life cycle or under various growth conditions. Different methods to measure pH(i) have been developed and among these methods, the use of pH-sensitive green fluorescent protein (GFP) as a pH(i) indicator is a promising technique. By using this approach, not only can more accurate pH(i) results be obtained but also long-term experiments on pH(i) can be performed. In this study, the wild type Zygosaccharomyces bailii, a notorious food spoilage yeast, was transformed with a plasmid encoding a pH-sensitive GFP (i.e. pHluorin), enabling the pH(i) of the yeast to be determined based on cellular fluorescent signals. After the transformation, growth and pH(i) of the yeast were investigated in four different acidic conditions at 22°C during 26days. From the experimental results, the transformation effectiveness was verified and a good correlation between yeast growth and pH(i) was noticed. Particularly, it was observed that the yeast has an ability to tolerate a significant pH(i) drop during exponential phase and a subsequent pH(i) recovery in stationary phase, which may underlie the exceptional acid resistance of the yeast. This was the first time that a GFP-based approach for pH(i) measurement was applied in Z. bailii and that the pH(i) of the yeast was monitored during such a long period (26days). It can be expected that greater understanding of the physiological properties and mechanisms behind the special acid resistance of the yeast will be obtained from further studies on this new yeast strain.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Zygosaccharomyces/fisiología , Ácidos/farmacología , Proteínas Fluorescentes Verdes/genética , Concentración de Iones de Hidrógeno , Plásmidos/genética , Zygosaccharomyces/crecimiento & desarrollo , Zygosaccharomyces/metabolismoRESUMEN
Growth/no growth (G/NG) studies that include the effect of medium structure have typically been performed for (pathogenic) bacteria and on the basis of gelatin/agar as a gelling agent. In this study, the growth potential of the spoilage yeast Zygosaccharomyces bailii was investigated in two model systems that resemble the macroscopic physicochemical and rheological properties of acidic sauces. In a Carbopol model system, the effect of pH (3.5-4.5), glycerol concentration (17-32%), acetic acid concentration (1.5-2.0%) and medium structure (3 levels) was investigated. In xanthan gum, the behavior of the yeast was studied at different levels of pH (3.5-4.5), NaCl concentration (0.5-13.5%), acetic acid concentration (0-2.0%) and medium structure (2 levels). Rheologically, viscoelastic moduli failed to discriminate between different forms of microbial growth, whereas yield stress data appeared to provide a better indication. In general, G/NG results revealed an unexpected increase of growth probability as a function of medium structure, both at 22 and 30 °C. Whether this behavior is the result of an underlying growth-promoting mechanism could not be explained from a macroscopic point of view (e.g., macrorheology, a(w)), but may be more related to the local microscopic properties of the gels. In a second part of this study, the potential use and information content of optical density measurements for G/NG data collection in structured media were critically evaluated and confronted with their practical relevance to the food industry.
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Microbiología de Alimentos/métodos , Polisacáridos Bacterianos/farmacología , Polivinilos/farmacología , Zygosaccharomyces/crecimiento & desarrollo , Resinas Acrílicas , Medios de Cultivo , Técnicas Microbiológicas/métodos , ReologíaRESUMEN
In a previous study on Zygosaccharomyces bailii, three growth/no growth models have been developed, predicting growth probability of the yeast at different conditions typical for acidified foods (Dang, T.D.T., Mertens, L., Vermeulen, A., Geeraerd, A.H., Van Impe, J.F., Debevere, J., Devlieghere, F., 2010. Modeling the growth/no growth boundary of Z. bailii in acidic conditions: A contribution to the alternative method to preserve foods without using chemical preservatives. International Journal of Food Microbiology 137, 1-12). In these broth-based models, the variables were pH, water activity and acetic acid, with acetic acid concentration expressed in volume % on the total culture medium (i.e., broth). To continue the previous study, validation experiments were performed for 15 selected combinations of intrinsic factors to assess the performance of the model at 22°C (60days) in a real food product (ketchup). Although the majority of experimental results were consistent, some remarkable deviations between prediction and validation were observed, e.g., Z. bailii growth occurred in conditions where almost no growth had been predicted. A thorough investigation revealed that the difference between two ways of expressing acetic acid concentration (i.e., on broth basis and on water basis) is rather significant, particularly for media containing high amounts of dry matter. Consequently, the use of broth-based concentrations in the models was not appropriate. Three models with acetic acid concentration expressed on water basis were established and it was observed that predictions by these models well matched the validation results; therefore a "systematic error" in broth-based models was recognized. In practice, quantities of antimicrobial agents are often calculated based on the water content of food products. Hence, to assure reliable predictions and facilitate the application of models (developed from lab media with high dry matter contents), it is important to express antimicrobial agents' concentrations on a common basis-the water content. Reviews over other published growth/no growth models in literature are carried out and expressions of the stress factors' concentrations (on broth basis) found in these models confirm this finding.
Asunto(s)
Antiinfecciosos/farmacología , Microbiología de Alimentos , Modelos Biológicos , Zygosaccharomyces/efectos de los fármacos , Zygosaccharomyces/crecimiento & desarrollo , Ácido Acético/farmacología , Medios de Cultivo , Concentración de Iones de Hidrógeno , Agua/metabolismoRESUMEN
BACKGROUND: Grass pollens are major triggers of allergic rhinitis and asthma, but the immunological relationships between pollen allergens of the subtropical Bahia grass, Paspalum notatum, and temperate grasses are unresolved. OBJECTIVE: To assess serum IgE cross-reactivity between subtropical P. notatum and temperate Lolium perenne (Ryegrass) pollen allergens. METHODS: Serum IgE reactivities of grass pollen-allergic patients with P. notatum, L. perenne and Cynodon dactylon (Bermuda grass) pollen extracts and their respective purified group 1 allergens, Pas n 1, Lol p 1 and Cyn d 1, were compared by immunoblotting, ELISA and basophil activation. RESULTS: In a cohort of 51 patients from a temperate region, a high frequency of IgE reactivity with each grass pollen was detected, but reactivity with L. perenne pollen was substantially greater than with P. notatum and C. dactylon pollen. Similarly, serum IgE reactivity with Lol p 1 was greater than with Pas n 1 or Cyn d 1. For seven of eight sera studied in detail, asymmetric serum IgE cross-reactivity was observed; L. perenne pollen inhibited IgE reactivity with P. notatum pollen but not the converse, and IgE reactivity with Pas n 1 was inhibited by Lol p 1 but IgE reactivity with Lol p 1 was not inhibited by Pas n 1 or Cyn d 1. Importantly, P. notatum pollen and Pas n 1 activated basophils in grass pollen-allergic patients from a temperate region, although stimulation was greater by pollen of L. perenne than P. notatum or C. dactylon, and by Lol p 1 than Pas n 1 or Cyn d 1. In contrast, a cohort of 47 patients from a subtropical region showed similar IgE reactivity with P. notatum and L. perenne pollen, and reciprocal cross-inhibition of IgE reactivity between L. perenne and P. notatum. CONCLUSIONS: Pollen allergens of the subtropical P. notatum, including Pas n 1, show clinically relevant IgE cross-reactivity with pollen allergens of L. perenne but also species-specific IgE reactivity.
Asunto(s)
Alérgenos/inmunología , Inmunoglobulina E/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Alérgenos/genética , Reacciones Cruzadas/inmunología , Cynodon/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/genética , Lolium/inmunología , Penicillium/inmunologíaRESUMEN
The aim of the study was to develop mathematical models describing growth/no growth (G/NG) boundaries of the highly resistant food spoilage yeast-Zygosaccharomyces bailii-in different environmental conditions, taking acidified sauces as the target product. By applying these models, the stability of products with characteristics within the investigated pH, a(w) and acetic acid ranges can be evaluated. Besides, the well-defined no growth regions can be used in the development of guidelines regarding formulation of new shelf-stable foods without using chemical preservatives, which would facilitate the innovation of additive-free products. Experiments were performed at different temperatures and periods (22 degrees C for 45 and 60days, 30 degrees C for 45days) in 150 modified Sabouraud media characterized by high amount of sugars (glucose and fructose, 15% (w/v)), acetic acid (0.0-2.5% (v/v), 6 levels), pH (3.0-5.0, 5 levels) and a(w) (0.93-0.97, 5 levels). These time and temperature combinations were chosen as they are commonly applied for shelf-stable foods. The media were inoculated with ca. 4.5 log CFU/ml and yeast growth was monitored daily using optical density measurements. Every condition was examined in 20 replicates in order to yield accurate growth probabilities. Three separate ordinary logistic regression models were developed for different tested temperatures and incubation time. The total acetic acid concentration was considered as variable for all models. In general, when one intrinsic inhibitory factor became more stringent, the G/NG boundary shifted to less stressful conditions of the other two factors, resulting in enlarged no growth zones. Abrupt changes of growth probability often occurred around the transition zones (between growth and no growth regions), which indicates that minor variations in environmental conditions near the G/NG boundaries can cause a significant impact on the growth probability. When comparing growth after 45days between the two tested temperatures, an unexpected phenomenon was observed: the no growth region at 30 degrees C was larger than the one at 22 degrees C, though it is known that 30 degrees C is the optimal growth temperature for Z. bailii. These results show that lowering temperature does not always lead to a reduced growth of the yeast (i.e. more stable foods) and storing shelf-stable products at the higher temperature (30 degrees C) is not always the worst case. In addition, at 22 degrees C, there was no significant difference in no growth zones between the two incubation periods (45 and 60days), implying that the no growth zones remain unchanged if the experimental time is sufficiently long.
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Microbiología de Alimentos , Conservación de Alimentos/métodos , Modelos Biológicos , Zygosaccharomyces/crecimiento & desarrollo , Ácido Acético , Medios de Cultivo , Conservantes de Alimentos , Tecnología de Alimentos , Concentración de Iones de Hidrógeno , Micología/métodos , Temperatura , AguaRESUMEN
Within the field of predictive microbiology, the number of studies that quantify the effect of food structure on microbial behavior is very limited. This is mainly due to impracticalities related to the use of a nonliquid growth medium. In this study, an experimental food model system for studying yeast spoilage in acid sauces was developed by selecting a suitable thickening/gelling agent. In a first step, a variety of thickening/gelling agents was screened, with respect to the main physicochemical (pH, water activity, and acetic acid and sugar concentrations) and rheological (weak gel viscoelastic behavior and presence of a yield stress) characteristics of acid sauces. Second, the rheological behavior of the selected thickening/gelling agent, Carbopol 980, was extensively studied within the following range of conditions: pH 4.0 to 5.0, acetic acid concentration of 0 to 1.0% (vol/vol), glycerol concentration of 0 to 15% (wt/vol), and Carbopol concentration of 1.0 to 1.5% (wt/vol). Finally, the applicability of the model system was illustrated by performing growth experiments in microtiter plates for Zygosaccharomyces bailii at 0, 0.5, 1.0, and 1.5% (wt/vol) Carbopol, 5% (wt/vol) glycerol, 0% (vol/vol) acetic acid, and pH 5.0. A shift from planktonic growth to growth in colonies was observed when the Carbopol concentration increased from 0.5 to 1.0%. The applicability of the model system was illustrated by estimating mu(max) at 0.5% Carbopol from absorbance detection times.
Asunto(s)
Ácidos , Microbiología de Alimentos , Tecnología de Alimentos/métodos , Modelos Teóricos , Zygosaccharomyces , Recuento de Colonia Microbiana , Manipulación de Alimentos , Modelos Lineales , Reología , Factores de Tiempo , Zygosaccharomyces/crecimiento & desarrolloRESUMEN
Stimulatory or protective effects of organic acids at low concentrations, e.g. acetic and lactic acid, on microorganisms have previously been reported. Especially in case of Zygosaccharomyces bailii, a peculiar growth stimulation by these two acids has recently been noticed. In order to elucidate this interesting phenomenon, growth and fermentative metabolism of Z. bailii was investigated in media with low pH (pH 4.0), high sugar (15% (w/v)) and different acetic and lactic acid concentrations. At both experimental temperatures (7 and 30 degrees C), a growth stimulation in the presence of 2.5% (v/v) lactic acid was observed. Furthermore at 7 degrees C, the yeast exhibited another unusual behaviour as it grew much faster in media containing 1.25% (v/v) acetic acid than in the control (without any acid). Production of fermentative metabolites was also increased together with the enhanced growth at both temperatures. These possible stimulatory effects of acetic and lactic acid should be taken into consideration when the acids are used at low doses for food preservative purpose. Presence of the acids may stimulate Z. bailii growth and fermentative metabolism, particularly at refrigeration temperature, consequently resulting in an earlier spoilage.
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Ácido Acético/farmacología , Contaminación de Alimentos/análisis , Ácido Láctico/farmacología , Zygosaccharomyces/efectos de los fármacos , Recuento de Colonia Microbiana , Relación Dosis-Respuesta a Droga , Fermentación , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Concentración de Iones de Hidrógeno , Temperatura , Zygosaccharomyces/crecimiento & desarrollo , Zygosaccharomyces/metabolismoAsunto(s)
Seguridad de Productos para el Consumidor , Microbiología de Alimentos , Conservación de Alimentos/métodos , Modelos Biológicos , Zygosaccharomyces/crecimiento & desarrollo , Recuento de Colonia Microbiana , Manipulación de Alimentos/métodos , Conservantes de Alimentos/efectos adversos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Matemática , Factores de Tiempo , Agua/metabolismoRESUMEN
Microbial spoilage of shelf-stable acidified sauces is predominantly caused by lactic acid bacteria and yeasts. A specific spoilage yeast in these products is Zygosaccharomyces bailii, as this fructophilic, osmotolerant, and weak acid resistant yeast is difficult to control. A growth/no growth model was developed describing the influence of (i) pH in a range from pH 3.0 to pH 5.0 (5 levels), (ii) acetic acid in a range from 0 to 3.5% (w/v), and (iii) lactic acid in a range from 0 to 3.0% (w/v). aw was fixed at a level of 0.95 which is representative for acidified sauces with high sugar content. Modified Sabouraud medium was inoculated at +/- 10(4) CFU/ml, incubated at 30 degrees C and growth was assessed by optical density measurements. All combinations of environmental conditions were tested in at least twelve replicates, yielding precise values for the probability of growth. Results showed that replacing acetic acid by lactic acid, which has a milder taste, may imply some risks on food spoilage because, under some conditions, stimulation of growth by lactic acid was observed. This stimulation had also consequences on the model development: (i) only ordinary logistic regression models were able to describe this phenomenon due to their flexible behaviour, (ii) it was necessary to split up the data set into two subsets to have the best description of the obtained data. Two different ordinary logistic regression models were fitted on these data sets taking either the total acid concentration as one of the explanatory variables or differentiating between the undissociated and dissociated acid concentrations. The obtained models were compared with the CIMSCEE code [CIMSCEE, 1992. Code for the production of microbiologically safe and stable emulsified and non-emulsified sauces containing acetic acid. Comité des Industries des Mayonnaise et Sauces Condimentaires, de la Communauté Economique Européenne, Brussels, Belgium], a formula which is nowadays often used by the food industry to predict the stability of acidified products based on the undissociated acetic acid, NaCl and sugars concentration. Comparing this formula and the newly developed models showed that the CIMSCEE code made a slight underestimation of the growth probability. Advantages of the newly developed models are the description of the gradual transition zone between growth and no growth and the incorporation of the effect of lactic acid, alone or in combination with acetic acid.
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Ácido Acético/farmacología , Contaminación de Alimentos/análisis , Ácido Láctico/farmacología , Modelos Biológicos , Agua/metabolismo , Zygosaccharomyces/crecimiento & desarrollo , Simulación por Computador , Relación Dosis-Respuesta a Droga , Contaminación de Alimentos/prevención & control , Concentración de Iones de Hidrógeno , Modelos Logísticos , Zygosaccharomyces/efectos de los fármacosRESUMEN
OBJECTIVES: This study aims to measure the private demand for oral cholera vaccines in Hue, Vietnam, an area of relatively low endemicity of cholera, using the contingent valuation method. METHODS: Interviews were conducted with either the head of household or spouse in 800 randomly selected households with children less than 18 years old. Respondents were asked whether they would purchase an oral cholera vaccine with different levels of effectiveness and durations of effectiveness (both for themselves and for other household members) at a specified price. RESULTS: The median respondent willingness to pay for 50% effective/3-year vaccine was estimated to be approximately $5, although 17% of the study sample would not pay for a cholera vaccine. The median economic benefit to a household of vaccinating all household members against cholera, as measured by its stated willingness to pay, was estimated to be $40 for a vaccine with these attributes. CONCLUSIONS: The perceived private economic benefits of a cholera vaccine were high, but not evenly distributed across the population. A minority of the people in Hue place no value on receiving a cholera vaccine.
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Actitud Frente a la Salud , Servicios de Salud del Niño/economía , Vacunas contra el Cólera/economía , Cólera/prevención & control , Prescripciones de Medicamentos/economía , Necesidades y Demandas de Servicios de Salud/estadística & datos numéricos , Adolescente , Adulto , Niño , Servicios de Salud del Niño/estadística & datos numéricos , Preescolar , Cólera/economía , Cólera/epidemiología , Vacunas contra el Cólera/provisión & distribución , Costo de Enfermedad , Composición Familiar , Femenino , Encuestas de Atención de la Salud , Accesibilidad a los Servicios de Salud , Necesidades y Demandas de Servicios de Salud/economía , Humanos , Entrevistas como Asunto , Masculino , Persona de Mediana Edad , Modelos Econométricos , Medición de Riesgo , Conducta de Reducción del Riesgo , Vietnam/epidemiologíaRESUMEN
These studies were undertaken to determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase (ADV/RSV-tk) and the prodrug ganciclovir (GCV) in an orthotopic murine bladder cancer model. We utilized a replication-defective adenoviral construct containing the beta-galactosidase gene as a control and the herpes simplex virus thymidine kinase gene as the therapeutic vector under the transcription control of the Rous sarcoma virus long terminal repeat promoter. Intravesically created, orthotopic bladder tumors were established in syngeneic C3H/He female mice. India ink injection and beta-galactosidase studies were performed to determine if transurethral administration, direct tumor injection, or the combination was the most efficient route of virus administration. Optimal dosing of ADV/RSV-tk was determined by direct tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate but similar controlled experiments. Growth curve studies demonstrated reliable tumor formation by 14 days. Direct transvesical tumor injection resulted in the best distribution and intratumor gene expression as measured by X-gal staining. Dose-ranging experiments demonstrated an optimal viral dose of 5 x 10(8) plaque-forming units and a greater than twofold reduction in tumor growth for the animals treated with ADV/RSV-tk compared to controls. Efficacy studies demonstrated a greater than threefold reduction in tumor growth. No clinical or gross pathologic toxicity was detected. Long-term survival results suggested a survival benefit for the treatment animals compared to controls. We conclude that ADV/RSV-tk in combination with GCV provides effective therapy for orthotopic murine bladder cancer by significantly inhibiting tumor growth with limited toxicity to the host. These data provide further support for testing this suicide gene therapy strategy in human Phase I trials.
Asunto(s)
Adenoviridae/genética , Terapia Genética , Neoplasias de la Vejiga Urinaria/terapia , Animales , Antivirales/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Ganciclovir/uso terapéutico , Vectores Genéticos , Herpesviridae/enzimología , Herpesviridae/genética , Humanos , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Timidina Quinasa/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Stromal-epithelial interactions in the prostate gland are dependent on androgen regulation of prostate stromal cells, yet little is known about androgen action in these cell types. Recent reports have demonstrated that androgen-regulated gene transcription can be stimulated or inhibited by certain growth factors, indicating cross-talk mechanisms. To address potential cross-talk in signaling pathways between androgen and transforming growth factor-beta1 (TGFbeta1) in prostate stromal cells, the PS-1 prostate smooth muscle cell line was examined. In the presence of physiological concentrations of androgen, PS-1 cell proliferation was stimulated, and androgen receptor (AR) exhibited a nuclear localization pattern. The addition of TGFbeta1 (25 pM) was capable of blocking androgen-induced proliferation, but had no direct effect in cultures without androgen. Immunocytochemistry to localize AR subcellular distribution showed that TGFbeta1 (5-100 pM) altered the distribution of AR from the nucleus to the cytoplasm. Other growth factors, including fibroblast growth factor-2, epidermal growth factor, and TGFbeta2 had no effect on AR distribution. The TGFbeta1-induced nuclear to cytoplasmic change in receptor localization was rapid (initiated within 30 min), was neutralized by TGFbeta1 antibodies, did not require new protein synthesis, and was complete by 6 h. Removal of TGFbeta1 from the culture medium resulted in a rapid redistribution of AR to the nucleus, indicating reversible mechanisms. Northern analysis of the ddp17 marker transcript for androgen action in PS-1 cells showed that androgen-stimulated ddp17 expression was inhibited in the presence of TGFbeta1 (25 pM). TGFbeta1 induced a similar nuclear to cytoplasmic distribution of AR in primary cultures of rat prostate stromal cells. TGFbeta1, however, had no effect on AR distribution in either the LNCaP prostatic carcinoma cell line or the DDT1MF-2 leiomyosarcoma cell line. Specific cross-talk between TGFbeta1 and AR signaling pathways in prostate stromal cells may play a significant role in prostate development and stromal cell response in carcinoma progression.
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Andrógenos/farmacología , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Próstata/efectos de los fármacos , Receptores Androgénicos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Línea Celular , Dihidrotestosterona/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Masculino , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Próstata/fisiología , Próstata/ultraestructura , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/ultraestructura , ARN Mensajero/metabolismo , Ratas , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Células Tumorales CultivadasRESUMEN
Transforming growth factor-beta1 (TGF-beta1) is implicated in prostate development, and elevated expression of TGF-beta1 has been correlated with prostate carcinogenesis. In this study, cell type specificity of TGF-beta1 and TGF-beta receptor Type II (RcII) protein expression was determined by immunocytochemistry in human normal prostate and compared to prostate carcinoma tissues. Heterogeneous localization patterns of LAP-TGF-beta1 (TGF-beta1 precursor) and RcII were observed in both epithelial and mesenchymal cells in fetal prostate, with LAP-TGF-beta1 localizing to more basal epithelial cells. Homogeneity of LAP-TGF-beta1 staining was increased in neonatal, prepubertal, and adult prostate, with elevated immunoreactivity noted in epithelial acini relative to stromal tissue for both LAP-TGF-beta1 and RcII proteins. In stromal tissues, RcII cell localization exhibited staining patterns nearly identical to smooth muscle alpha-actin. In prostate carcinoma, LAP-TGF-beta1 localized to carcinoma cells with an increased staining heterogeneity relative to normal prostate. In contrast to normal epithelial cells, carcinoma epithelial cells exhibited low to nondetectable RcII staining. Stromal cell staining patterns for LAP-TGF-beta1 and RcII in carcinoma, however, were identical to those of normal prostate stromal cells. These studies implicate both epithelial and stromal cells as sites of TGF-beta1 synthesis and RcII localization in the developing and adult normal human prostate. In addition, these data indicate a loss of epithelial expression of RcII concurrent with altered LAP-TGF-beta1 expression in human prostate carcinoma cells.
Asunto(s)
Fragmentos de Péptidos , Próstata/química , Neoplasias de la Próstata/química , Precursores de Proteínas , Receptores de Factores de Crecimiento Transformadores beta/análisis , Factor de Crecimiento Transformador beta/análisis , Adolescente , Adulto , Niño , Humanos , Inmunohistoquímica , Lactante , Masculino , Próstata/embriología , Próstata/crecimiento & desarrollo , Proteínas Serina-Treonina Quinasas , Proteínas/análisis , Receptor Tipo II de Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta1RESUMEN
We previously reported the purification of ps20 (Rowley, D. R., Dang, T. D., Larsen, M., Gerdes, M. J., McBride, L., and Lu, B. (1995) J. Biol. Chem. 270, 22058-22065), a urogenital sinus mesenchymal cell secreted protein having growth-inhibitory properties. We report here cloning of the 1.03-kilobase rat ps20 cDNA clone from the PS-1 (adult rat prostate smooth muscle) cDNA library. Partial clones were obtained by nested polymerase chain reaction with degenerate primers, and full-length ps20 cDNA clones were isolated by plaque hybridization. Sequence analysis revealed that ps20 protein contains a WAP-type "four-disulfide core" motif and is a novel member of the WAP signature protein family composed primarily of secreted serine protease inhibitors. Native ps20 immunoprecipitated from smooth muscle cells and recombinant ps20 both resolved on SDS-polyacrylamide gel electrophoresis with apparent molecular mass of 27-29 kDa under reducing conditions and 21-23 kDa under non-reducing conditions, respectively. Stable ps20-transfectant COS-7 cell lines secreted ps20 and were growth-inhibited relative to mock transfectants. In addition, COS-7 and prostate carcinoma PC-3 cells were growth-inhibited by bacterially expressed ps20. Northern analysis indicated differential expression by tissue with highest expression in the heart. Immunohistochemical localization of ps20 protein showed cell-specific expression by both visceral and vascular smooth muscle in all tissues, including the prostate gland. These results indicate ps20 is a novel growth-regulatory member of the WAP signature family expressed by smooth muscle cells.
Asunto(s)
Disulfuros/química , Inhibidores de Crecimiento/genética , Músculo Liso/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , División Celular , Línea Celular , Clonación Molecular , ADN Complementario , Femenino , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Músculo Liso/citología , Proteínas/química , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVES: To determine the feasibility, safety, and efficacy of suicide gene therapy using adenoviral-mediated herpes simplex virus thymidine kinase gene (HSV-tk) and the prodrug ganciclovir (GCV) in a murine model of human transitional cell carcinoma. METHODS: We used a replication-defective adenoviral construct containing the beta-galactosidase gene (ADV/Rous sarcoma virus [RSV]-beta-gal) as a control or ADV/RSV-tk as the therapeutic vector under the transcriptional control of the RSV long-terminal repeat promoter. Transduction efficiency was assessed in vitro by infection of MBT-2 cells with ADV/RSV-beta-gal at various multiplicities of infection (MOI) utilizing 5-bromo-4-chlor-3-indolyl-beta-D-galactoside (X-gal) staining. Sensitivity of MBT-2 cells to the therapeutic vector was determined after infection with ADV/RSV-tk with or without GCV. Subcutaneous tumors were established in syngeneic C3H/He female mice with 5 x 10(5) MBT-2 cells. Optimal dosing of ADV/RSV-tk was determined by direct percutaneous tumor injection with increasing viral doses and treatment with GCV. Treatment efficacy, long-term survival, and toxicity were determined in separate, similar, controlled experiments. RESULTS: In vitro studies indicated greater than 95% transduction 96 hours after inoculation at an MOI of 3000 and a greater than 95% cell death rate with RSV-tk + GCV at an MOI of 61 or greater. In vivo experiments demonstrated an optimal viral dose of 3 x 10(8) plaque-forming units (pfu) and a greater than fourfold reduction in tumor growth for the animals treated with ADV/RSV-tk compared with control animals (P = 0.0013). Toxicity was limited to histologic evidence of hepatitis with ADV/RSV-tk doses greater than 3 x 10(8) pfu + GCV. Long-term survival of treatment animals was significantly increased over that of control animals (59%, P = 0.0001). CONCLUSIONS: ADV/RSV-tk with GCV treatment results in efficient gene transfer in vitro and provides effective therapy in experimental murine bladder cancer by significantly inhibiting tumor growth and improving host survival.
Asunto(s)
Adenoviridae/genética , Carcinoma de Células Transicionales/terapia , Terapia Genética/métodos , Vectores Genéticos , Neoplasias de la Vejiga Urinaria/terapia , Animales , Estudios de Factibilidad , Ganciclovir , Ratones , Ratones Endogámicos C3H , Simplexvirus/genética , Timidina Quinasa/genéticaRESUMEN
Androgens play a key role in directing stromal-epithelial interactions in prostate gland development, in maintenance of adult phenotype, and in disease progression. To address the molecular mechanisms of androgen action in prostate stromal cells and the genes regulated by androgens in this cell type, a stromal cell line (PS-1) was developed from rat ventral prostate. The PS-1 cell line was adapted to chemically defined media and characterized as smooth muscle based on expression of desmin, smooth muscle alpha-actin, and nuclear androgen receptor, markers for prostate smooth muscle. To examine steroid hormone regulation, PS-1 cells were analyzed for response to a panel of steroid hormones in serum-free, chemically defined media. PS-1 cells were significantly growth stimulated by physiological concentrations of androgens (10nM) relative to other steroids tested. To ascertain whether this cell line could be used to examine androgen-regulated gene expression, differential display PCR was used to demonstrate the presence of androgen-regulated transcripts and provide the initial steps in cloning these genes. Replicate differential display PCR analyses showed consistent androgen stimulation of 16 messenger RNA species. Northern analysis confirmed androgen regulation of 6 species. Sequence analysis of each indicated no regions of significant homology or direct matches to existing sequences. Further study of the PS-1 stromal cell line and androgen-regulated genes identified here will provide a novel in vitro system for defining molecular mechanisms of androgen action in prostate stromal cells and the significance of stromal androgen-regulated genes to stromal-epithelial interactions.
Asunto(s)
Andrógenos/farmacología , Próstata/citología , Animales , División Celular/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica , Masculino , Próstata/metabolismo , Ratas , Ratas Sprague-Dawley , Transcripción Genética/efectos de los fármacosRESUMEN
Our previous studies have characterized mesenchyme-derived proteins to identify biologically active proteins and novel markers for stromal cell paracrine action relative to stromal-epithelial interactions. Previous reports have characterized properties of a growth inhibitory activity (to bladder and prostatic epithelial cells), secreted by U4F fetal rat urogenital sinus mesenchymal cells, not cross-reactive with antibodies to known cytokines, and provisionally termed UGIF. The present study reports the characterization, purification, and biological properties of a 20-21-kDa protein responsible for UGIF activity. The 20-21-kDa protein (termed ps20) was purified to near homogeneity, the amino-terminal sequence was determined, and biological properties were characterized in vitro. Amino-terminal sequence analysis indicated no direct matches or regions of homology with known proteins. Purified ps20 induced a linear and saturable inhibition of [3H]thymidine incorporation in PC-3 prostatic carcinoma cells (half-maximal activity at 2.6 nM), inhibited cell proliferation (increased population doubling time from 19.8 to 25.8 h), and induced a 210% stimulation in the synthesis of secreted proteins. These data suggest that ps20 may be a candidate paracrine effector protein and may play a role in stromal-epithelial cell interaction in the prostate gland.