RESUMEN
Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.
Asunto(s)
Enfermedades Transmitidas por los Alimentos , Moringa oleifera , Infecciones Estafilocócicas , Animales , Staphylococcus aureus/genética , Pseudomonas aeruginosa/genética , Leche , Moringa oleifera/genética , Enterotoxinas/genética , Enterotoxinas/metabolismo , Enterotoxinas/farmacología , Microbiología de Alimentos , Antibacterianos/farmacología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/veterinaria , Biopelículas , Enfermedades Transmitidas por los Alimentos/veterinaria , Expresión GénicaRESUMEN
Currently, Clostridium perfringens enterotoxin (CPE) is being investigated as an anti-cancer drug for tumors expressing the tight junction (TJ) transmembrane proteins claudin-3 and/or claudin-4. However, the optimal conditions for CPE cytotoxicity are still unclear. Our objectives were to determine the optimal conditions for CPE as an anti-cancer drug for treating ovarian cancer in vitro and in vivo. In our experiments, cells at low culture density showed higher sensitivity to CPE, suggesting that claudins at TJs were poorly accessible to CPE compared with those at the edge of cell colonies. Ovarian cancer cells cultured under calcium-depleted pretreatment conditions to disrupt TJs and to knock-down TJ proteins and E-cadherin production altered CPE cytotoxicity, which was mainly dependent on claudin-4 expression. These results suggest that the condition of claudin-4 at the cell surface is important for CPE cytotoxicity. Our in vivo experiments showed that a high dose of CPE is required for the effective treatment of peritoneal dissemination of ovarian cancer cells. Here, we suggest that the accessibility of CPE to claudins is important for its cytotoxicity and depends on the conditions of claudin-4 in vitro. In addition, E-cadherin expression in ovarian cancer cells affects the efficiency of CPE in vivo.
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Antineoplásicos/farmacología , Claudina-4/genética , Enterotoxinas/farmacología , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Claudina-3/genética , Claudina-3/metabolismo , Claudina-4/antagonistas & inhibidores , Claudina-4/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule's immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate-labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.
Asunto(s)
Claudina-4/metabolismo , Enterotoxinas/farmacología , Tioestreptona/farmacología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Animales , Células CACO-2 , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Impedancia Eléctrica , Transferencia Resonante de Energía de Fluorescencia , Ensayos Analíticos de Alto Rendimiento , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Masculino , Ratas Wistar , Proteínas Recombinantes/metabolismoRESUMEN
Many reports have shown that crude extracts of the American cockroach have therapeutic effects on inflammation. In a previous study, our research group showed that an antimicrobial peptide (Periplanetasin-2) derived from the American cockroach via de novo transcriptome analysis inhibited apoptosis of human colonocytes and inflammatory responses of the mouse gut caused by Clostridium difficile toxin A. Here, we examined whether Periplanetasin-4 (Peri-4), another antimicrobial peptide identified via de novo transcriptome analysis of the American cockroach, could also inhibit the various toxicities induced by C. difficile toxin A. We found that Peri-4 significantly reduced the cell viability loss and cell apoptosis caused by toxin A in vitro. Peri-4 also ameliorated the severe inflammatory responses seen in the toxin A-induced mouse enteritis model, rescuing the villus disruption and interleukin-6 production induced by luminal injection of toxin A into the mouse gut. Mechanistically, we found that Peri-4 could reduce toxin A-induced reactive oxygen species production to inhibit the activations of p38MAPK and p21Cip1/Waf1 , which are critical for the cell damages induced by toxin A. These results collectively suggest that the Peri-4 may be a potential therapeutic agent for treating toxin A-induced pseudomembranous colitis. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
Asunto(s)
Antiinflamatorios/farmacología , Toxinas Bacterianas/antagonistas & inhibidores , Enteritis/tratamiento farmacológico , Enterotoxinas/antagonistas & inhibidores , Proteínas de Insectos/farmacología , Animales , Toxinas Bacterianas/farmacología , Evaluación Preclínica de Medicamentos , Enteritis/inmunología , Enteritis/metabolismo , Enterotoxinas/farmacología , Células HT29 , Humanos , Íleon/efectos de los fármacos , Íleon/inmunología , Íleon/patología , Ratones , Periplaneta/química , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Distraction osteogenesis (DO) technique could be used to manage large-size bone defect successfully, but DO process usually requires long duration of bone consolidation. Innovative approaches for augmenting bone consolidation are of great need. Staphylococcal enterotoxin C2 (SEC2) has been found to suppress osteoclastogenesis of mesenchymal stem cells in vitro. In this study, we investigated the effect of SEC2 on proliferation and osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). Further, we locally administrated SEC2 (10 ng/ml) or PBS into the distraction gap in Sprague-Dawley male rat DO model every 3 days till termination at 3 and 6 weeks. The regenerates were subjected to X-rays, micro-computed tomography, mechanical testing, histology, and immunohischemistry examinations to assess new bone quality. SEC2 had no effect on cell viability. The calcium deposition was remarkably increased and osteogenic marker genes were significantly up-regulated in rBMSCs treated with SEC2. In rat DO model, SEC2 group had higher bone volume/total tissue volume in the regenerates. At 6 weeks, mechanical properties were significantly higher in SEC2-treated tibiae comparing to the control group. Histological analysis confirmed that the new bone had improved quality in SEC2 treated group, where the osteocalcin and osterix expression in the regenerates was up-regulated, indicating faster bone formation. The current study demonstrated that SEC2 local injection promotes osteogenesis and enhanced bone consolidation in DO. The findings support application of SEC2 as a potential novel strategy to expedite bone consolidation in patients undergoing DO treatment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1215-1225, 2017.
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Células de la Médula Ósea/efectos de los fármacos , Enterotoxinas/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis por Distracción , Osteogénesis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Módulo de Elasticidad , Enterotoxinas/uso terapéutico , Interleucinas/sangre , Masculino , Cultivo Primario de Células , Ratas Sprague-Dawley , Microtomografía por Rayos XRESUMEN
The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and phosphorylation status of EphA2 and Src after C-CPE treatment and found changes in expression and phosphorylated status of these regulatory molecules. These observations show that after additional preclinical studies CPE and C-CPE in combinations with other drugs can be considered as a potential modalities for intravesical treatment of BC because of its ability to effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium.
Asunto(s)
Antineoplásicos/farmacología , Claudina-4/genética , Clostridium perfringens/química , Enterotoxinas/farmacología , Células Epiteliales/efectos de los fármacos , Administración Intravesical , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Claudina-4/metabolismo , Dasatinib/farmacología , Evaluación Preclínica de Medicamentos , Enterotoxinas/biosíntesis , Enterotoxinas/aislamiento & purificación , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Mitomicina/farmacología , Modelos Biológicos , Terapia Molecular Dirigida , Unión Proteica , Receptor EphA2/genética , Receptor EphA2/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Urotelio/efectos de los fármacos , Urotelio/metabolismo , Urotelio/patología , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
BACKGROUND: Isoflavone-containing soy products modulate allergic inflammation in mice. In our previously study, IFN-γ and IL-10 production increased in mice fed with Saccharomyces cerevisiae legume fermented product (SCLFP), demonstrating that SCLFP had immunomodulatory activity. In this study, we tested the anti-inflammatory effects of SCLFP in a mouse model of cutaneous atopic dermatitis inflammation induced by epicutaneous sensitization. METHODS: Epicutaneous exposure to protein allergens plus Staphylococcal enterotoxin B induced a T helper (Th)-2-dominant immune response as well as cutaneous atopic dermatitis-like inflammation in BALB/c mice. The thickness of the skin epithelium, eosinophil migration, and T helper responses were determined in patched skin and draining lymph nodes of mice fed with and without SCLFP. RESULTS: Epicutaneous exposure to protein allergens plus Staphylococcal enterotoxin B induced a T helper (Th)-2-dominant immune response as well as cutaneous atopic dermatitis-like inflammation in BALB/c mice. SCLFP feeding attenuated this cutaneous Th2 response, as evidenced by decreased thickening of the epidermis, less eosinophil infiltration, and lower levels of IL-5, IL-13, and CXCL11 expression compared to controls. Oral administration of SCLFP also modulated Th1 responses in draining lymph nodes, with lower levels of IFN-γ, IL-4, and IL-17 expression. CONCLUSION: Oral intake of SCLFP modulated the induced Th2 inflammatory responses in skin and might have potential applications for the prevention and treatment of atopic dermatitis.
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Dermatitis Atópica/tratamiento farmacológico , Fabaceae/metabolismo , Factores Inmunológicos/farmacología , Extractos Vegetales/farmacología , Saccharomyces cerevisiae/metabolismo , Alérgenos/inmunología , Alérgenos/metabolismo , Animales , Dermatitis Atópica/inmunología , Dermatitis Atópica/metabolismo , Modelos Animales de Enfermedad , Enterotoxinas/inmunología , Enterotoxinas/farmacología , Fabaceae/química , Fabaceae/microbiología , Femenino , Fermentación , Factores Inmunológicos/química , Factores Inmunológicos/inmunología , Factores Inmunológicos/metabolismo , Interleucinas/inmunología , Interleucinas/metabolismo , Ratones , Ratones Endogámicos BALB C , Extractos Vegetales/química , Extractos Vegetales/inmunología , Extractos Vegetales/metabolismo , Piel/inmunología , Piel/metabolismo , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
The BCR-ABL kinase inhibitor imatinib is highly effective in the treatment of chronic myeloid leukemia (CML). However, long-term imatinib treatment induces immunosuppression, which is mainly due to T cell dysfunction. Imatinib can reduce TCR-triggered T cell activation by inhibiting the phosphorylation of tyrosine kinases such as Lck, ZAP70, LAT, and PLC γ 1 early in the TCR signaling pathway. The purpose of this study was to investigate whether the superantigen SEA, a potent T cell stimulator, can block the immunosuppressive effects of imatinib on T cells. Our data show that the exposure of primary human T cells and Jurkat cells to SEA for 24 h leads to the upregulation of the Lck and ZAP70 proteins in a dose-dependent manner. T cells treated with SEA prior to TCR binding had increased the tyrosine phosphorylation of Lck, ZAP70, and PLC γ 1. Pretreatment with SEA prevents the inhibitory effects of imatinib on TCR signaling, which leads to T cell proliferation and IL-2 production. It is conceivable that SEA antagonizes the imatinib-mediated inhibition of T cell activation and proliferation through the TCR signaling pathway.
Asunto(s)
Benzamidas/antagonistas & inhibidores , Benzamidas/farmacología , Enterotoxinas/farmacología , Piperazinas/antagonistas & inhibidores , Piperazinas/farmacología , Pirimidinas/antagonistas & inhibidores , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Mesilato de Imatinib , Células Jurkat , Fosforilación , Proteínas Tirosina Quinasas/análisis , Proteínas Tirosina Quinasas/metabolismoRESUMEN
BACKGROUND: The effect of labour and different labour-related factors on the cord blood (CB) cell cytokine production is still relatively unknown. OBJECTIVE: To study the relationships between the production of IL-5, IL-10 and IFN-γ in CB samples and maternal, early neonatal and birth-related factors. METHODS: Whole-blood samples were collected after birth (n=423) and they were stimulated for 24 and 48 h with a combination of phorbol ester and ionomycin. Production of IL-5, IL-10 and IFN-γ was determined using ELISA. Maternal, early neonatal and birth-related variables were recorded prospectively during pregnancy, and during and after delivery. RESULTS: After multivariable adjustment for confounders, the strongest predictor of IL-5, IL-10 and IFN-γ production in CB cell samples was the season of birth. Children born in the spring had significantly lower cytokine responses compared with those born in the fall. IL-5 production was inversely associated with female gender of the child and maternal smoking. If corrections for white blood cell (WBC) counts were not performed, IL-5 production was also significantly associated with the mode of delivery. Respectively, the production of IL-10 and IFN-γ was inversely associated with prostaglandin induction before birth. CONCLUSION: Environmental exposure to pollen and ultraviolet irradiation during gestation may have an effect on the cytokine profile of the offspring in CB because children born in the spring or winter showed the lowest IL-5, IL-10 and IFN-γ responses. The production of IL-10 and IFN-γ was also inversely associated with prostaglandin labour induction before birth. Other labour-related factors were not significantly associated with production of IL-5, IL-10 and IFN-γ after WBC count correction.
Asunto(s)
Células Sanguíneas/inmunología , Sangre Fetal/inmunología , Interferón gamma/sangre , Interleucina-10/sangre , Interleucina-5/sangre , Estaciones del Año , Células Sanguíneas/efectos de los fármacos , Células Sanguíneas/efectos de la radiación , Distribución de Chi-Cuadrado , Parto Obstétrico/métodos , Enterotoxinas/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Sangre Fetal/citología , Finlandia , Humanos , Ionomicina/farmacología , Recuento de Leucocitos , Leucocitosis/inmunología , Lipopolisacáridos/farmacología , Masculino , Polen/inmunología , Embarazo , Estudios Prospectivos , Prostaglandinas/uso terapéutico , Medición de Riesgo , Factores de Riesgo , Factores Sexuales , Fumar/efectos adversos , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Rayos UltravioletaRESUMEN
The epithelial barrier of the intestine and the gut-associated lymphoid tissue (GALT) protects the host against luminal pathogenic micro-organisms. This is important at weaning, when animals are exposed to infectious agents and stresses. We have developed a rat model of intestinal inflammation post weaning, based on the systemic administration of Staphylococcus aureus enterotoxin B (SEB). Since the inflammatory response obtained is mild, the food intake pattern is not affected, which makes this model useful for studies of nutritional therapies for intestinal inflammatory disease. SEB increased T-lymphocytes in Peyer's patches and the number of activated T-lymphocytes in mesenteric lymph nodes (organized GALT). In the lamina propria, SEB increased activated T-lymphocytes as well as cytotoxic and natural killer-cell populations of the diffuse GALT. It also increased pro-inflammatory cytokines and inflammatory mediators in both Peyer's patches and mucosa. Rats given SEB had higher paracellular permeability to macromolecules, which was associated with a reduction in epithelial tightness. This model was used to examine whether dietary supplementation with spray-dried animal plasma proteins affects intestinal inflammation. Results showed that dietary plasma proteins can attenuate the mucosal immune response in both organized and diffuse GALT and that these effects are mediated by a reduction in the production of pro-inflammatory cytokines.
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Antiinflamatorios/uso terapéutico , Proteínas Sanguíneas/uso terapéutico , Citocinas/metabolismo , Enterotoxinas/farmacología , Inflamación/tratamiento farmacológico , Mucosa Intestinal/efectos de los fármacos , Staphylococcus aureus , Animales , Antiinflamatorios/farmacología , Proteínas Sanguíneas/farmacología , Proteínas en la Dieta/administración & dosificación , Suplementos Dietéticos , Modelos Animales de Enfermedad , Inmunidad Mucosa , Inflamación/inducido químicamente , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Ganglios Linfáticos Agregados/efectos de los fármacos , Ganglios Linfáticos Agregados/metabolismo , Ratas , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca(2+) as the intracellular messenger. Similarly, extracellular Ca(2+) (Ca(2+)(o)) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca(2+)(o). Here, preconditioning with ST, mediated by GCC signaling through cyclic nucleotide-gated channels, restored Ca(2+)(o)-dependent cytostasis, reflecting posttranscriptional regulation of calcium-sensing receptors (CaRs). ST-induced GCC signaling deployed CaRs to the surface of human colon cancer cells, whereas elimination of GCC signaling in mice nearly abolished CaR expression in enterocytes. Moreover, ST-induced Ca(2+)(o)-dependent cytostasis was abrogated by CaR-specific antisense oligonucleotides. Importantly, following ST preconditioning, newly expressed CaRs at the cell surface represented tumor cell receptor targets for antiproliferative signaling by CaR agonists. Since expression of the endogenous paracrine hormones for GCC is uniformly lost early in carcinogenesis, these observations offer a mechanistic explanation for the Ca(2+)(o)-resistant phenotype of colon cancer cells. Restoration of antitumorigenic CaR signaling by GCC ligand replacement therapy represents a previously unrecognized paradigm for the prevention and treatment of human colorectal cancer employing dietary Ca(2+) supplementation.
Asunto(s)
División Celular/efectos de los fármacos , Neoplasias del Colon/patología , Enterotoxinas/farmacología , Receptores Sensibles al Calcio/fisiología , Apoptosis , Línea Celular Tumoral , Transformación Celular Neoplásica , Neoplasias Colorrectales/patología , Humanos , Precondicionamiento Isquémico , Necrosis , Receptores Sensibles al Calcio/efectos de los fármacosRESUMEN
Authors contributed equally to this manuscript Natural adjuvants, such as bacterial lipopolysaccharide (LPS), activate antigen presenting cells via Toll-like receptors and, indirectly, increase the survival of antigen-activated T cells. The molecular mechanisms leading to increased survival remain poorly defined. Because T cell clonal expansion leads to high energy demands, we hypothesized that increased glucose uptake and/or utilization in adjuvant-activated T cells could be important molecular event(s) that would lead to adjuvant-associated T cell survival advantage. Using a fluorescent analog of 2-deoxyglucose, 2-NBDG, we measured glucose accumulation and rate of uptake in T cells from mice treated with antigen in the absence or presence of LPS. Although adjuvant activated T cells increased the accumulation of 2-NBDG, the rate of uptake was unchanged compared to cells activated with only antigen. Furthermore, glucose transport inhibitors, cytochalasin B or phloretin, decreased the accumulation of glucose in adjuvant-treated T cells, but this decrease did not impair adjuvant-associated survival advantages. Together, these data indicate that increased glucose uptake through glucose transporters is not required for increased survival of activated T cells.
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Adyuvantes Inmunológicos/farmacología , Glucosa/metabolismo , Activación de Linfocitos/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocalasina B/farmacología , Relación Dosis-Respuesta a Droga , Enterotoxinas/farmacología , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucosa/análisis , Glucosa/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Mediciones Luminiscentes , Ratones , Ratones Endogámicos , Floretina/farmacología , Factores de TiempoRESUMEN
BACKGROUND: A reduction in microbial burden during infancy when allergen-specific memory is evolving has become a prominent explanation for the allergy epidemic. OBJECTIVE: We sought to determine whether probiotic dietary supplementation in the first 6 months of life could modify allergen- and vaccine-specific immune responses. METHODS: Two hundred and thirty-one pregnant women with a history of allergic disease and positive allergen skin prick test (SPT) were recruited into a randomized-controlled trial. The infants received either a probiotic (3 x 10(9)Lactobacillus acidophilus LAVRI-A1; Probiomics) or placebo (maltodextrin alone) daily for the first 6 months of life, given independent of feeding methods. One hundred and seventy-eight children completed the study; blood samples were available from 60 children in the placebo group and 58 children in the probiotic group. Infant cytokine (IL-5, IL-6, IL-10, IL-13, TNF-alpha or TGF-beta) responses to tetanus toxoid (TT), house dust mite (HDM), ovalbumin (OVA), beta-lactoglobulin (BLG), Staphylococcus enterotoxin B (SEB) and phytohaemaglutinin (PHA) were measured at 6 months of age. RESULTS: Children who received the probiotics showed reduced production of IL-5 and TGF-beta in response to polyclonal (SEB) stimulation (P=0.044 and 0.015, respectively). They also demonstrated significantly lower IL-10 responses to TT vaccine antigen compared with the placebo group (P=0.03), and this was not due to any differences in vaccination. However, there were no significant effects of probiotics on either Type 1 (Th1) or Type 2 (Th2) T helper cell responses to allergens or other stimuli. The only other effects observed were for reduced TNF-alpha and IL-10 responsiveness to HDM allergens in children receiving probiotics (P=0.046 and 0.014, respectively). CONCLUSIONS: In summary, although we did not see any consistent effects on allergen-specific responses, our study suggests that probiotics may have immunomodulatory effects on vaccine responses. The significance and clinical relevance of this need to be determined in further studies.
Asunto(s)
Alérgenos/inmunología , Suplementos Dietéticos , Hipersensibilidad/prevención & control , Lactobacillus acidophilus , Probióticos/administración & dosificación , Vacunas/inmunología , Antígenos Dermatofagoides/farmacología , Células Cultivadas , Enterotoxinas/farmacología , Humanos , Hipersensibilidad/inmunología , Lactante , Recién Nacido , Interleucina-10/inmunología , Interleucina-5/inmunología , Lactoglobulinas/farmacología , Leucocitos Mononucleares/inmunología , Neutrófilos/inmunología , Ovalbúmina/farmacología , Fitohemaglutininas/farmacología , Toxoide Tetánico/farmacología , Células TH1/inmunología , Células Th2/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Bacterial superantigens (SAg) are potent T cell activators and when delivered systemically elicit a self-limiting enteropathy in mice. Also, SAg-stimulated human peripheral blood mononuclear cells (PBMC) increase enteric epithelial cell monolayer permeability in vitro. Epigallocatechin gallate (EGCG), the major polyphenol component of green tea (Camilla sinesis) leaf, has been presented as an anti-inflammatory agent. We tested the hypothesis that EGCG (10-100 microM) would block PBMC activation by the SAg, Staphylococcus aureus enterotoxin B (SEB, 1 microg/ml), thus preventing disruption of the epithelial barrier. Pretreatment or co-treatment of human PBMC or murine lymphnode cells with EGCG significantly reduced SEB-induced proliferation and IL-2, IFNgamma, and TNFalpha production. ConA-induced proliferation was also inhibited by EGCG (50 microM) co-treatment. These effects of EGCG were not due to induction of immune cell apoptosis, and were independent of EGCGs anti-oxidant activity, and inhibition of NF-kappaB or AP-1 activation. Moreover, addition of exogenous IL-2 (20 ng/ml) to the cultures could not overcome the immunosuppressive effect of EGCG. Culture supernatant from PBMC stimulated in the presence of EGCG failed to increase the permeability of T84 epithelial cell monolayers: a finding consistent with the reduced IFNgamma and TNFalpha production by SAg+EGCG treated PBMC. These data promote EGCG as a suppressor of T cell activation, and given the prominent role that bacteria and T cells play in inflammatory disease we suggest that EGCG could be a useful addition to current treatments for enteric immune disorders and T cell driven immunopathologies.
Asunto(s)
Antiinflamatorios/farmacología , Catequina/análogos & derivados , Enterotoxinas/farmacología , Células Epiteliales/efectos de los fármacos , Linfocitos/efectos de los fármacos , Animales , Camellia sinensis , Catequina/farmacología , Proliferación Celular/efectos de los fármacos , Citocinas/efectos de los fármacos , Citocinas/inmunología , Ensayo de Cambio de Movilidad Electroforética , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Humanos , Immunoblotting , Interleucina-2/biosíntesis , Interleucina-2/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfocitos/inmunología , Ratones , FN-kappa B/inmunología , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , Factor de Transcripción AP-1/inmunología , Factor de Transcripción AP-1/metabolismoRESUMEN
Proinflammatory cytokines mediate the toxic effects of superantigenic staphylococcal exotoxins (SE) and bacterial lipopolysaccharide (LPS). Triptolide, an oxygenated diterpene derived from a traditional Chinese medicinal herb, Tripterygium wilfordii, inhibited SE-stimulated T-cell proliferation (by 98%) and expression of interleukin 1beta, interleukin 6, tumor necrosis factor, gamma interferon, monocyte chemotactic protein 1, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta by human peripheral blood mononuclear cells (PBMC). It also blocked the production of these cytokines and chemokines by LPS-stimulated PBMC in a dose-dependent manner. These results suggest that triptolide has potent immunosuppressive effects even counteracting the effects of superantigens and LPS. It also may be therapeutically useful for mitigating the pathogenic effects of these microbial products by downregulating the signaling pathways activated by both bacterial exotoxins and endotoxins.
Asunto(s)
Toxinas Bacterianas/farmacología , Proliferación Celular/efectos de los fármacos , Citocinas/biosíntesis , Diterpenos/farmacología , Medicamentos Herbarios Chinos/farmacología , Endotoxinas/farmacología , Enterotoxinas/farmacología , Fenantrenos/farmacología , Superantígenos/farmacología , Linfocitos T/efectos de los fármacos , Toxinas Bacterianas/antagonistas & inhibidores , Células Cultivadas , Quimiocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Endotoxinas/antagonistas & inhibidores , Enterotoxinas/antagonistas & inhibidores , Compuestos Epoxi , Humanos , Staphylococcus aureus/inmunología , Linfocitos T/citología , Linfocitos T/metabolismoRESUMEN
The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues. The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)). We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin [KLH] and hen egg lysozyme [HEL]). Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH. Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB. The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent. The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch. We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/farmacología , Enterotoxinas/inmunología , Enterotoxinas/farmacología , Proteínas de Escherichia coli , Inmunidad Mucosa/efectos de los fármacos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , ADP Ribosa Transferasas/química , Adyuvantes Inmunológicos/química , Administración Intranasal , Administración Oral , Animales , Toxinas Bacterianas/química , Toxina del Cólera/farmacología , Enterocitos/efectos de los fármacos , Enterocitos/inmunología , Enterotoxinas/química , Hemocianinas , Inmunización , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/químicaRESUMEN
BACKGROUND: Surgical resection of pancreatic tumors removes gross disease but not metastases. Adjuvant therapy such as chemotherapy and radiation treatment is of little value in metastatic pancreatic cancer. The hypothesis of this investigation is that specific and effective immunotherapeutic vaccine (dendritic/tumor cell fusion) will activate cytotoxic T lymphocytes (CTLs), leading to the eradication of spontaneous pancreatic cancer. METHODS: We have developed a double transgenic mouse model (MET) that forms spontaneous pancreatic tumors and expresses the human MUC1 antigen. Seven-week-old MET mice (n = 8) were treated every 3 weeks with the vaccine. In addition, these mice received 50 microg of superantigen staphylococcal enterotoxin B (SEB), a known T cell stimulant, prior to the first vaccination. A second treatment group received SEB alone (n = 8) and controls received no treatment (n = 9). MUC1-specific CTLs were measured by chromium release assay. At 10 weeks of age and at necropsy, MUC1 serum levels were measured using a MUC1-specific ELISA. RESULTS: Mice were known to harbor microscopic foci of cancer at birth. Survival was enhanced in vaccine as well as SEB-treated mice (75% CI +/- 0.42) compared to controls (11% CI +/- 0.28) and both groups of treated mice exhibited mature CTLs without in vitro stimulation. MUC1 serum levels of the vaccine group were 50% less than that of control (P < 0.04) at 10 weeks. MUC1 serum levels directly correlated with tumor weight at necropsy (r = 0.86). CONCLUSIONS: This is the first evidence that MUC1-specific CTLs can be stimulated to enhance survival in a spontaneous tumor model.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Fusión Celular , Células Dendríticas/inmunología , Enterotoxinas/farmacología , Neoplasias Pancreáticas/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Modelos Animales de Enfermedad , Inmunización , Inmunoterapia , Ratones , Ratones Endogámicos C57BL , Mucina-1/sangre , Mucina-1/inmunología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Células Tumorales CultivadasRESUMEN
The non-toxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent mucosal adjuvant and immunomodulator capable of blocking autoimmune disease. These effects are linked with its ability to modulate lymphocyte populations--a feature that is dependent on binding to ubiquitously expressed cell surface receptors. Here, we demonstrate that EtxB can trigger up-regulated expression of class II MHC and CD25 on purified populations of B lymphocytes, suggesting that EtxB can directly activate biochemical signalling pathways in these cells. The nature of the intracellular signalling events was investigated. B cells cultured with EtxB, but not a non-receptor binding mutant protein, EtxB(G33D), caused the activation of the extracellular signal-regulated kinase (Erk) forms of mitogen-activated protein (MAP) kinase in a process that was dependent on MAPK/Erk kinase (MEK), phosphoinositide 3-kinase (PI3-kinase) and protein kinase C (PKC), as determined by the use of specific inhibitors. PI3-kinase was critical not only in the activation of MAP kinase but also in the up-regulation of both class II and CD25. However, MEK inhibition only partially abrogated the EtxB-mediated up-regulation of MHC class II expression and did not affect CD25 expression--findings suggesting that additional pathways downstream of PI3-kinase are involved. A role for PKC in these processes was suggested by the finding that inhibitors of PKC completely blocked EtxB-mediated CD25 up-regulation. Thus, we have shown that receptor binding by EtxB triggers multiple signalling pathways in B cells that regulate the expression of key cell surface molecules.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Toxinas Bacterianas/farmacología , Enterotoxinas/farmacología , Proteínas de Escherichia coli , Animales , Antígenos de Superficie/metabolismo , Linfocitos B/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Escherichia coli/inmunología , Femenino , Técnicas In Vitro , Activación de Linfocitos/efectos de los fármacos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteína Quinasa C/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
We studied the effects of the phosphodiesterase inhibitors pentoxifylline (PTX) and rolipram (ROL) on nitric oxide (NO) production by macrophages and correlated this with cellular cAMP levels. The RAW 264.7 cell line or mouse peritoneal macrophages were activated with lipopolysaccharide (LPS) and interferon gamma (IFN gamma), with or without ROL, PTX, cAMP analogues, or Forskolin. In vivo, peritoneal macrophages were stimulated with staphylococcal enterotoxin B with or without administration of ROL. Nitrite levels in culture and the total cellular cAMP levels were measured. ROL and PTX suppressed NO production of LPS/IFN gamma-stimulated macrophages. ROL (IC(50) = 68-74 microM) was about 40 times more potent than PTX (IC(50) = 2.4-2.9 mM). The suppression paralleled increased total cellular cAMP level (EC(50) = 68-72 microM) and was mimicked by other cAMP elevating agents. ROL and PTX suppressed inducible NO synthase at the mRNA level. The inhibition of NO production of macrophages by ROL or PTX could be beneficial in NO-mediated inflammatory and/or autoimmune disorders.