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BACKGROUND AND OBJECTIVES: Tunneled cuffed hemodialysis catheters (TCC) get colonized by microorganisms, increasing risk for catheter related bacteremia (CRB). Our objective was to detect the prevalence of bacterial colonization of TCC by using quantitative PCR (qPCR) targeting 16S rRNA and by determining the intraluminal adherent biological material (ABM) coverage. METHODS: A total of 45 TCC were investigated. The 16S rRNA qPCR technique was used to detect bacterial colonization after scraping the intraluminal ABM. Proximal, middle, and distal TCC were evaluated by scanning electron microscopy (SEM) to determine the percentage (%) of intraluminal ABM coverage. All catheters were cultured following sonication. RESULTS: A total of 45 TCC were removed: 7 due to CRB, 3 for suspected CRB and 35 were removed for non-infectious etiologies. Bacterial colonization was detected in 27 TCC by documenting 16S rRNA qPCR (+) results (60%). Seven of these 16S rRNA qPCR (+) catheters were removed due to CRB. There was no difference in demographic, clinical, or laboratory values between the 16S rRNA (+) versus (-) TCC. The 16S rRNA qPCR (-) outcome was highly associated with CRB-free status with negative predictive value of 100%. Bacterial colonization was documented in 10 TCC using catheter cultures (22%), which was significantly less compared to qPCR method (p = 0.0002). ABM were detected in all catheter pieces, with mean intraluminal surface coverage (ABMC) of 68.4 ± 26.1%. ABM was unlikely to be microbial biofilm in at least 36% of removed TCC as their 16S rRNA qPCR and catheter culture results were both negative. CONCLUSIONS: Detecting bacterial colonization of TCC was significantly higher with 16S rRNA qPCR compared to catheter cultures. The 16S rRNA qPCR (-) cannot be predicted and was strongly associated with absence of CRB. Intraluminal ABM was not associated with microbial presence in about 1/3 of the TCC. These pieces of evidence may help to improve prophylactic strategies against CRB.
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Bacteriemia , Diálisis Renal , Bacteriemia/diagnóstico , Catéteres/efectos adversos , Catéteres de Permanencia/efectos adversos , Humanos , Microscopía Electrónica de Rastreo , Reacción en Cadena de la Polimerasa , Prevalencia , ARN Ribosómico 16S/genética , Diálisis Renal/efectos adversos , Diálisis Renal/métodosRESUMEN
The FRUVEDomics study investigates the effect of a diet intervention focused on increasing fruit and vegetable intake on the gut microbiome and cardiovascular health of young adults with/at risk for metabolic syndrome (MetS). It was hypothesized that the recommended diet would result in metabolic and gut microbiome changes. The 9-week dietary intervention adhered to the US Department of Agriculture Dietary Guidelines for Americans and focused on increasing fruit and vegetable intake to equal half of the diet. Seventeen eligible young adults with/or at high risk of MetS consented and completed preintervention and postintervention measurements, including anthropometric, body composition, cardiovascular, complete blood lipid panel, and collection of stool sample for microbial analysis. Participants attended weekly consultations to assess food logs, food receipts, and adherence to the diet. Following intention-to-treat guidelines, all 17 individuals were included in the dietary, clinical, and anthropometric analysis. Fruit and vegetable intake increased from 1.6 to 3.4 cups of fruits and vegetables (Pâ¯<â¯.001) daily. Total fiber (Pâ¯=â¯.02) and insoluble fiber (Pâ¯<â¯.0001) also increased. Clinical laboratory changes included an increase in sodium (Pâ¯=â¯.0006) and low-density lipoprotein cholesterol (Pâ¯=â¯.04). In the fecal microbiome, Erysipelotrichaceae (phylum Firmicutes) decreased (log2 fold change: -1.78, Pâ¯=â¯.01) and Caulobacteraceae (phylum Proteobacteria) increased (log2 fold changeâ¯=â¯1.07, Pâ¯=â¯.01). Implementing a free-living 9-week diet, with intensive education and accountability, gave young adults at high risk for/or diagnosed with MetS the knowledge, skills, and feedback to improve diet. To yield greater impact, a longer diet intervention may be needed in this population.
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Dieta/métodos , Frutas , Síndrome Metabólico/dietoterapia , Educación del Paciente como Asunto/métodos , Verduras , Adulto , Femenino , Humanos , Masculino , Adulto JovenRESUMEN
The 2010 USDA Dietary Guidelines for Americans (DGA) recommends a diet largely composed of fruit and vegetables. Consuming a diet high in fruit and vegetables and low in refined carbohydrates and saturated fat may reduce an individual's risk for type 2 diabetes, nonalcoholic fatty liver disease, low-grade chronic inflammation, and metabolic syndrome (MetS). Several recent studies have implicated the bioactive sphingolipid ceramide as an associative and causative biomarker for the development of these conditions. Considering that the intake of fruit and vegetables is frequently inadequate in young adults, we performed a pilot investigation to assess the efficacy of a free-living fruit and vegetable intervention on overall metabolic health, circulating ceramide supply, and inflammatory status in young adults. We discovered that adoption of the recommended DGA for fruit and vegetable intake for 8 weeks decreased waist circumference, systolic blood pressure, and circulating cholesterol. Lipidomics analysis revealed that nutritional intervention can lower circulating ceramides, including C24:0 ceramide, a known inhibitor of insulin signaling. Unexpectedly, we observed an increase in C16:0 ceramide, suggesting that this form of ceramide in circulation is not associated with metabolic disease in humans. We also observed an improved inflammatory status with enhanced fruit and vegetable intake that was correlated with ceramide concentrations. These data suggest that adopting the recommended DGA is associated with a reduction of many, but not all, ceramide species and may help to prevent or mitigate MetS. Future research needs to assess whether the ceramide-lowering ability of nutritional intervention is associated with reduced risk of developing metabolic disease.
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Ceramidas/sangre , Dieta/métodos , Frutas , Síndrome Metabólico/prevención & control , Verduras , Adolescente , Adulto , Presión Sanguínea , Colesterol/sangre , Dieta/efectos adversos , Dietoterapia/efectos adversos , Dietoterapia/métodos , Femenino , Humanos , Masculino , Síndrome Metabólico/dietoterapia , Política Nutricional , Proyectos PilotoRESUMEN
ß-Elemene, originally derived from plants, has been recently investigated as a new anticancer agent. The purpose of this study was to explore the efficacy and mechanisms of action of the combined use of ß-elemene plus a taxane as an antitumor therapeutic strategy for ovarian cancer and other carcinomas. The interaction of ß-elemene with paclitaxel or docetaxel produced additive to moderately synergistic effects against the platinum-resistant ovarian cancer cell line A2780/CP70 and its parental cell line A2780, and showed moderately synergistic activity against PC-3 prostate cancer cells. In addition, the co-administration of ß-elemene and a taxane at low-micromolar concentrations dramatically increased the rate of micronucleus formation and the percentage of mitotic arrest in both ovarian cancer cell lines, as compared with treatment with either agent alone. The highest synergy towards the ovarian cancer cells was observed with ß-elemene plus docetaxel. Consistent with these data, treatment of A2780/CP70 cells with ß-elemene plus a taxane strikingly reduced cell viability and increased cell apoptosis, as assessed by annexin V binding. Moreover, ß-elemene plus docetaxel induced elevated levels of caspase-9 and p53 proteins in A2780/CP70 cells, and the combination of ß-elemene plus a taxane caused marked cell-cycle arrest at the G2/M phase in these cells. One possible mechanism to account for the enhanced cytotoxic efficacy of this combination treatment is a ß-elemene-induced increase in taxane influx into cancer cells. These observations indicate that combination therapy with ß-elemene and taxanes has synergistic antitumor activity against ovarian and prostate carcinomas in vitro. This promising new therapeutic combination warrants further pre-clinical exploration for the treatment of chemoresistant ovarian cancer and other types of tumors.
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Antineoplásicos Fitogénicos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Sesquiterpenos/farmacología , Taxoides/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Docetaxel , Sinergismo Farmacológico , Femenino , Humanos , Neoplasias Ováricas/patología , Paclitaxel/farmacologíaRESUMEN
The development of effective agents for overcoming platinum chemoresistance in lung carcinoma continues to have high priority. We have demonstrated recently that ß-elemene, a novel antitumor compound, enhances cisplatin activity by triggering lung cancer cell death via apoptosis. Here, we investigated whether ß-elemene acts synergistically with cisplatin to inhibit non-small cell lung cancer (NSCLC) cell proliferation by blocking cell cycle progression. ß-Elemene substantially increased the suppressive effect of cisplatin on cell growth and proliferation in the NSCLC cell lines H460 and A549. Furthermore, ß-elemene augmented cisplatin in the cell cycle arrest of NSCLC cells at G(2)/M. This was associated with upregulated checkpoint kinase (CHK2) expression and reduced CDC2 activity (i.e., increased phosphorylation of CDC2 on Tyr-15 and decreased phosphorylation of CDC2 on Thr-161). Moreover, ß-elemene and cisplatin in combination clearly decreased the protein levels of cyclin B1 and CDC25C and increased the levels of p21(Cip1/Waf1), p27(Kip1), and GADD45 in these cells, compared with the effects of either agent alone at the same concentration. These results suggest that the ß-elemene-enhanced inhibitory effect of cisplatin on lung carcinoma cell proliferation is regulated by a CHK2-mediated CDC25C/CDC2/cyclin B1 signaling pathway and leads to the blockade of cell cycle progression at G(2)/M. A comparison of the cytotoxic efficacies of ß-elemene and three synthetic analogs (ß-elemenol, ß-elemenal, and ß-elemene fluoride) in the two lung cancer cell lines revealed that ß-elemenol and ß-elemene fluoride had the same antitumor efficacy as ß-elemene, whereas ß-elemenal was appreciably more potent than ß-elemene. Thus, although all three synthetic analogs of ß-elemene considerably suppressed NSCLC cell growth and proliferation, ß-elemenal may have greater potential as an anticancer alternative to ß-elemene in treating lung cancer and other tumors.
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Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Cisplatino/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Sesquiterpenos/farmacología , Proteína Quinasa CDC2 , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa de Punto de Control 2 , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
BACKGROUND: To investigate the potential association between oral health and cognitive function, a pilot study was conducted to evaluate high throughput DNA sequencing of the V3 region of the 16S ribosomal RNA gene for determining the relative abundance of bacterial taxa in subgingival plaque from older adults with or without dementia. METHODS: Subgingival plaque samples were obtained from ten individuals at least 70 years old who participated in a study to assess oral health and cognitive function. DNA was isolated from the samples and a gene segment from the V3 portion of the 16S bacterial ribosomal RNA gene was amplified and sequenced using an Illumina HiSeq1000 DNA sequencer. Bacterial populations found in the subgingival plaque were identified and assessed with respect to the cognitive status and oral health of the participants who provided the samples. RESULTS: More than two million high quality DNA sequences were obtained from each sample. Individuals differed greatly in the mix of phylotypes, but different sites from different subgingival depths in the same subject were usually similar. No consistent differences were observed in this small sample between subjects separated by levels of oral health, sex, or age; however a consistently higher level of Fusobacteriaceae and a generally lower level of Prevotellaceae was seen in subjects without dementia, although the difference did not reach statistical significance, possibly because of the small sample size. CONCLUSIONS: The results from this pilot study provide suggestive evidence that alterations in the subgingival microbiome are associated with changes in cognitive function, and provide support for an expanded analysis of the role of the oral microbiome in dementia.
RESUMEN
BACKGROUND: West Virginia has the worst oral health in the United States, but the reasons for this are unclear. This pilot study explored the etiology of this disparity using culture-independent analyses to identify bacterial species associated with oral disease. METHODS: Bacteria in subgingival plaque samples from twelve participants in two independent West Virginia dental-related studies were characterized using 16S rRNA gene sequencing and Human Oral Microbe Identification Microarray (HOMIM) analysis. Unifrac analysis was used to characterize phylogenetic differences between bacterial communities obtained from plaque of participants with low or high oral disease, which was further evaluated using clustering and Principal Coordinate Analysis. RESULTS: Statistically different bacterial signatures (P<0.001) were identified in subgingival plaque of individuals with low or high oral disease in West Virginia based on 16S rRNA gene sequencing. Low disease contained a high frequency of Veillonella and Streptococcus, with a moderate number of Capnocytophaga. High disease exhibited substantially increased bacterial diversity and included a large proportion of Clostridiales cluster bacteria (Selenomonas, Eubacterium, Dialister). Phylogenetic trees constructed using 16S rRNA gene sequencing revealed that Clostridiales were repeated colonizers in plaque associated with high oral disease, providing evidence that the oral environment is somehow influencing the bacterial signature linked to disease. CONCLUSIONS: Culture-independent analyses identified an atypical bacterial signature associated with high oral disease in West Virginians and provided evidence that the oral environment influenced this signature. Both findings provide insight into the etiology of the oral disparity in West Virginia.
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Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Enfermedades de la Boca/microbiología , Filogenia , ARN Ribosómico 16S/genética , Enfermedades Dentales/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Análisis por Conglomerados , Placa Dental/microbiología , Humanos , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Proyectos Piloto , Análisis de Componente Principal , West Virginia/epidemiología , Adulto JovenRESUMEN
IgE-mediated food allergies are an important cause of life-threatening hypersensitivity reactions. Orally administered peanut antigens mixed with the mucosal adjuvant cholera toxin (CT) induce a strong peanut extract (PE)-specific serum IgE response that is correlated with T-helper type 1 (Th1) and type 2 (Th2)-like T-cell responses. This study was conducted to determine if respiratory enteric orphan virus (reovirus), a non-pathogenic virus that induces robust Th1-mediated mucosal and systemic responses could modulate induction of PE-specific allergic responses when co-administered with PE. Young mice were orally exposed to PE mixed with CT, reovirus, or both CT and reovirus. As expected, CT promoted PE-specific serum IgE, IgG1, and IgG2a and intestinal IgA production as well as splenic Th1- and Th2-associated cytokine recall responses. Reovirus did not alter PE-specific serum IgE and IgG1 levels, but substantially increased the PE-specific IgG2a response when co-administered with PE with or without CT. Additionally, reovirus significantly decreased the percentage of the Peyer's patch CD8+ T-cells and Foxp3+CD4+ T-regulatory cells when co-administered with PE. These results demonstrate that an acute mucosal reovirus infection and subsequent Th1 immune response is capable of modulating the Th1/Th2 controlled humoral response to PE. The reovirus-mediated increase in the PE-specific IgG2a antibody response may have therapeutic implications as increased levels of non-allergenic PE-specific IgG2a could block PE antigens from binding to IgE-sensitized mast cells.
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Arachis/inmunología , Inmunoglobulina G/inmunología , Hipersensibilidad al Cacahuete/inmunología , Infecciones por Reoviridae/inmunología , Animales , Arachis/química , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Línea Celular , Células Cultivadas , Toxina del Cólera/inmunología , Toxina del Cólera/farmacología , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina A/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Intestino Delgado/efectos de los fármacos , Intestino Delgado/inmunología , Intestino Delgado/virología , Ratones , Ratones Endogámicos C3H , Ganglios Linfáticos Agregados/inmunología , Ganglios Linfáticos Agregados/virología , Extractos Vegetales/inmunología , Extractos Vegetales/farmacología , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Células TH1/efectos de los fármacos , Células TH1/inmunología , Técnicas de Cultivo de TejidosRESUMEN
Cisplatin is one of the most potent chemotherapeutic agents for the treatment of many types of solid tumours. Nevertheless, it is not the first-line drug for prostate cancer chemotherapy, because prostate tumour cells exhibit intrinsic and acquired resistance to cisplatin. We have previously demonstrated that ß-elemene, a novel plant-derived anti-neoplastic with low toxicity, inhibits lung and ovarian carcinoma cell growth in vitro. In the present study, we explored the therapeutically chemosensitizing effect of ß-elemene on cisplatin anti-tumour efficacy in androgen-independent prostate cancer cells as well as the underlying mechanism. ß-Elemene significantly increased cisplatin cytotoxicity in the androgen-independent prostate carcinoma cell lines DU145 and PC-3. In addition, ß-elemene markedly promoted cisplatin-induced apoptotic cell death in both cell lines, as determined by three different apoptosis assays. ß-Elemene augmented the cisplatin-induced activation of caspase-3/7/10 and caspase-9, cleavage of caspase-3 and -9, suppression of Bcl-2 and Bcl-X(L) expression, and release of cytochrome c from mitochondria in these cells. Thus, ß-elemene enhancement of cisplatin-induced apoptosis via mitochondrial activation of the caspase-mediated apoptotic pathway may account for the augmented anti-cancer potency of cisplatin in prostate cancer. Cisplatin combined with ß-elemene as a chemosensitizer or adjuvant warrants further study and may be potentially useful as a first-line treatment of androgen-independent prostate carcinomas.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Western Blotting , Caspasas/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Cisplatino/administración & dosificación , Cisplatino/farmacología , Cisplatino/uso terapéutico , Sinergismo Farmacológico , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Etiquetado Corte-Fin in Situ , Masculino , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Sesquiterpenos/administración & dosificación , Sesquiterpenos/farmacología , Sesquiterpenos/uso terapéuticoRESUMEN
Acetaminophen (APAP)-induced hepatotoxicity accounts for nearly half of acute liver failure cases in the United States. The doses that produce hepatotoxicity vary considerably and many risk factors have been proposed, including liver inflammation from viral hepatitis. Interestingly, inflammatory stress from another stimulus, bacterial endotoxin (lipopolysaccharide, LPS), renders the liver more sensitive to hepatotoxicity from numerous xenobiotic agents. The purpose of these studies was to test the hypothesis that inflammation induced by LPS or infection with reovirus increases sensitivity to APAP-induced liver injury. For LPS-induced inflammation, C57BL/6J mice were treated with either saline or LPS (44 x 10(6) EU/kg, ip) 2 h before treatment with APAP (100-400 mg/kg, ip) or saline. No elevation in serum alanine aminotransferase (ALT) activity was observed in mice that received vehicle or LPS alone. LPS co-treatment produced a leftward shift of the dose-response curve for APAP-induced hepatotoxicity and led to significantly greater tumor necrosis factor-alpha (TNF) production than APAP alone. Reovirus serotype 1 (10(8) PFU, iv) induced inflammation in Balb/c mice as evidenced by increases in hepatic mRNAs for macrophage inhibitory protein-2, interleukin-6, and TNF. Co-administration of reovirus and APAP at doses of 450 and 700 mg/kg (2 h after reovirus) led to increases in serum ALT activity, whereas neither reovirus nor APAP alone produced liver injury. Consistent with the increases in serum ALT activity, histopathologic examination revealed centrilobular necrosis with marked neutrophilic accumulation only in livers of mice treated with LPS/APAP or with reovirus/APAP. The results suggest that normally noninjurious doses of APAP are rendered hepatotoxic by modest inflammation, whether bacterial or viral in origin.
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Acetaminofén/efectos adversos , Hígado/efectos de los fármacos , Infecciones por Reoviridae/inmunología , Animales , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Glutatión/metabolismo , Inflamación/inducido químicamente , Inflamación/microbiología , Inflamación/virología , Lipopolisacáridos/farmacología , Hígado/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neutrófilos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
beta-Elemene, a new plant-derived anticancer agent with low toxicity, has been reported to be effective in the treatment of leukemia and solid tumors. In the current study, we explored the therapeutic application of beta-elemene in sensitizing lung cancer cells to cisplatin. beta-Elemene considerably enhanced the inhibitory effect of cisplatin on cell proliferation in a time- and dose-dependent manner in the human non-small cell lung cancer (NSCLC) cell lines H460 and A549. Furthermore, this effect of beta-elemene on cisplatin activity occurred through the induction of apoptosis in NSCLC cells, as assessed by an ELISA-based assay, TUNEL assay and annexin V binding assay. Consistent with these results, the protein levels of Bax and phospho-Bcl-2 increased and those of Bcl-2 and XIAP decreased in cells treated with beta-elemene in combination with cisplatin, compared with the levels in cells treated with either agent alone. Finally, beta-elemene augmented the cisplatin-induced increases in caspase-3, -7, -9 and -10 activities and cleaved caspase-3, -9 and poly(ADP-ribose) polymerase levels in NSCLC cells. These observations suggest that beta-elemene sensitizes NSCLC cells to cisplatin via a mitochondria-mediated intrinsic apoptosis pathway involving Bcl-2 family proteins and IAPs (inhibitor of apoptosis proteins). Our data provide a rationale for developing a combination of beta-elemene and cisplatin as a regimen for the treatment of lung carcinoma and other cisplatin-resistant tumors.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Antineoplásicos Fitogénicos/administración & dosificación , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Caspasas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Neoplasias Pulmonares/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sesquiterpenos/administración & dosificación , Factores de Tiempo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Beta-elemene has recently raised interest in P.R. China as a novel antitumor plant drug isolated from the Chinese medicinal herb Zedoary. To explore potentially useful combinations of beta-elemene with taxanes in the clinic, we characterized the effects of beta-elemene combined with taxanes in human lung cancer cells using a median effect analysis, micronucleus assay, apoptotic detection, and determination of gene expression in the signaling pathways of apoptosis. The synergistic analysis indicated that the interactions of beta-elemene with paclitaxel or docetaxel ranged from slight synergism to synergism. Combinations of beta-elemene with docetaxel induced much stronger synergistic interactions in p53 mutant H23 cells and p53 null H358 cells than in p53 wild-type H460 and A549 cells. Similar synergistic interactions were observed by micronucleus assay, apoptotic detection, and determination of apoptotic gene expression. Our findings indicate that the synergistic effects achieved with combinations of beta-elemene and taxanes are related to the augmented cytotoxic efficacy of taxanes owing to the action of beta-elemene. In H460 and A549 cells, dose-dependent upregulation of p53 protein expression was observed in cultures treated with docetaxel alone and with docetaxel plus beta-elemene, whereas no significant change in p53 expression was observed in any of the treatment groups in H23 cells. Fas revealed no alteration of expression with any of the treatments in this study. However, the combination treatments induced increased cytochrome c release from mitochondria, significant caspase-8 and -3 cleavage, and downregulation of Bcl-2 and Bcl-XL expression. These results suggest that, although p53 plays an important role in taxane-induced cell death, apoptosis induced by beta-elemene or in combination with docetaxel thereof seems to be initiated through a p53- and Fas-independent pathway via mitochondria in our lung cancer cells. The suppression of specific 'survival' gene expression appears to be the key action leading to the synergistic effect of combination treatments with beta-elemene and taxanes. Finally, the beta-elemene-induced alteration of cell membrane permeability, which has potential to result in enhanced cellular uptake of taxanes, may also contribute to the synergistic interactions of the combination treatments.
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Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/tratamiento farmacológico , Sesquiterpenos/administración & dosificación , Taxoides/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Genes p53 , Humanos , Técnicas In Vitro , Concentración 50 Inhibidora , Medicina Tradicional China , Mutación , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
The trichothecene mycotoxin deoxynivalenol (DON), a frequent contaminant of cereal grains, is known to dysregulate mucosal and systemic immunity. In this study, we tested the hypothesis that DON interferes with the murine immune response to viral respiratory infection. Female Balb/c mice (5 weeks old) were orally gavaged with DON (10 mg/kg body weight [bw]) or saline vehicle and then intranasally instilled with 10(7) plaque-forming units of reovirus serotype 1, strain Lang (T1/L). At 10-day postinstillation (PI), both viral titers and reovirus L(2) gene expression were 10-fold higher in lungs of DON-treated mice than in saline controls. The lowest observed effective DON dose that impaired viral clearance was 2 mg/kg bw. Although DON amplified reovirus-induced interferon (IFN)-beta and IFN-gamma mRNA responses in lung, the toxin suppressed mRNA expression for IFN-alpha, IFN-alphabeta receptor (IFNAR), and IFN-gamma receptor (IFNGR). DON also impaired induction of two type 1 IFN-dependent antiviral genes, double-stranded RNA activated protein kinase R (PKR) and oligoadenylate synthase 2 (OAS2). Respiratory reovirus infection caused a mild bronchopneumonia in mice which was markedly exacerbated by DON as evidenced by severe inflammatory cell infiltration, marked alveolar damage, and a higher volume density of intraepithelial mucosubstances in pulmonary airways. At 3- and 7-day PI, elevations in total protein, MCP-1, TNF-alpha, total cells, macrophages, neutrophils, and lymphocytes were observed in bronchoalveolar lavage fluid (BALF) of control mice infected with reovirus. DON markedly enhanced viral-induced elevations of protein, MCP-1, TNF-alpha, and inflammatory cells in the BALF at 3-day PI. DON exposure also upregulated induction of reovirus-specific immunoglobulin A (IgA) in BALF, fecal pellets, and serum. DON's effect on BALF IgA was preceded by elevated IL-6 expression and secretion in the lung. Taken together, the results suggest that DON compromised resistance to respiratory viral infection. Reduced expression of IFNAR and type 1 IFN-mediated genes in the lung might contribute to DON impairment of pulmonary reovirus clearance, whereas exacerbation of bronchopneumonia and IgA responses corresponded to increased MCP-1, TNF-alpha, and IL-6 expression.
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Bronconeumonía , Pulmón , Orthoreovirus de los Mamíferos/aislamiento & purificación , Infecciones por Reoviridae , Tricotecenos/toxicidad , Animales , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Bronconeumonía/inmunología , Bronconeumonía/virología , Femenino , Genes Virales/genética , Inmunidad Mucosa/efectos de los fármacos , Inmunoglobulina A/inmunología , Pulmón/patología , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Orthoreovirus de los Mamíferos/genética , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Respiratory enteric orphan virus (reovirus) has been used to study many aspects of the biology and genetics of viruses, viral infection, pathogenesis, and the immune response to virus infection. This report describes the functional activity of virus labeled with Alexa Fluor 488, a stable fluorescent dye. Matrix assisted laser desorption-time of flight analysis indicated that Alexa Fluor 488 labeled the outer capsid proteins of reovirus. Labeled virus bound to murine L929 fibroblasts as determined by flow cytometry and fluorescence microscopy, and the specificity of binding were demonstrated by competitive inhibition with non-labeled virus. Labeled reovirus induced apoptosis and cytopathic effect in infected L929 cells. Mice infected with labeled virus mounted robust serum antibody and CD8(+) T-cell responses, indicating that labeled virus retained immunogenicity in vivo. These results indicate that Alexa Fluor 488-labeled virus provides a powerful new tool to analyze reovirus infection in vitro and in vivo.
Asunto(s)
Orthoreovirus Mamífero 3/química , Infecciones por Reoviridae/inmunología , Coloración y Etiquetado/métodos , Succinimidas/química , Virión/química , Animales , Proteínas de la Cápside/química , Colorantes Fluorescentes/química , Masculino , Orthoreovirus Mamífero 3/inmunología , Orthoreovirus Mamífero 3/patogenicidad , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BLRESUMEN
Trichothecenes are exquisitely toxic to the gastrointestinal (GI) tract and leukocytes and thus are likely to impair gut immunity. The purpose of this research was to test the hypothesis that the Type A trichothecene T-2 toxin interferes with the gut mucosal immune response to enteric reovirus infection. Mice were exposed i.p. first to 1.75 mg/kg bw T-2 and then 2 h later with 3 x 10(7) plaque-forming units of reovirus serotype 1, strain Lang (T1/L). As compared to vehicle-treated control, T-2-treated mice had dramatically elevated intestinal plaque-forming viral titers after 5 days and failed to completely clear the virus from intestine by 10 days. Levels of reovirus lambda2 core spike (L2 gene) RNA in feces in T-2-treated mice were significantly higher at 1, 3, 5, and 7 days than controls. T-2 potentiated L2 mRNA expression in a dose-dependent manner with as little as 50 microg/kg of the toxin having a potentiative effect. T-2 exposure transiently suppressed induction of reovirus-specific IgA in feces (6 and 8 days) as well as specific IgA and IgG2a in serum (5 days). This suppression corresponded to decreased secretion of reovirus-specific IgA and IgG2a in Peyer's patch (PP) and lamina propria fragment cultures prepared 5 days after infection. T-2 suppressed IFN-gamma responses in PP to reovirus at 3 and 7 days as compared to infected controls whereas IL-2 mRNA concentrations were unaffected. PP IL-6 mRNA levels were increased 2-fold 2 h after T-2 treatment, but no differences between infected T-2-exposed and infected vehicle-treated mice were detectable over the next 7 days. Overall, the results suggest that T-2 toxin increased both the extent of GI tract reovirus infection and fecal shedding which corresponded to both suppressed immunoglobulin and IFN-gamma responses.
Asunto(s)
Enfermedades Gastrointestinales/inmunología , Inmunoglobulinas/biosíntesis , Interferón gamma/biosíntesis , Orthoreovirus de los Mamíferos/crecimiento & desarrollo , Infecciones por Reoviridae/inmunología , Toxina T-2/toxicidad , Animales , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Heces/virología , Enfermedades Gastrointestinales/virología , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/inmunología , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inmunoglobulinas/inmunología , Interferón gamma/inmunología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/virología , Ratones , Infecciones por Reoviridae/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esparcimiento de Virus/efectos de los fármacosRESUMEN
Polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA and IgA-coated immune complexes from the lamina propria across epithelia and into secretions. The effect of reovirus infection on regulation of pIgR expression in the human intestinal epithelial cell line HT-29 was characterized in this report. Both replication-competent and UV-inactivated reovirus at m.o.i. equivalents of 1-100 p.f.u. per cell upregulated pIgR mRNA by 24 h post-infection and intracellular pIgR protein was increased at 48 h following exposure to UV-inactivated virus. Binding of virus to HT-29 cells was required, as pre-incubating virus with specific antiserum, but not non-immune serum, inhibited reovirus-mediated pIgR upregulation. Endosomal acidification leading to uncoating of virus is a required step for pIgR upregulation, as ammonium chloride or bafilomycin A1 pre-treatment inhibited virus-induced pIgR upregulation. Inhibition experiments using the calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal suggested that calpains are involved in reovirus-mediated pIgR upregulation. Upregulation of pIgR following virus infection appears to be an innate immune response against invading pathogens that could help the host clear infection effectively. Signalling induced by microbes and their products may serve to augment pIgR-mediated transcytosis of IgA, linking the innate and acquired immune responses to viruses.
Asunto(s)
Mucosa Intestinal/metabolismo , Orthoreovirus Mamífero 3/inmunología , Receptores de Inmunoglobulina Polimérica/metabolismo , Calpaína/antagonistas & inhibidores , Calpaína/fisiología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Mucosa Intestinal/virología , Leupeptinas/farmacología , Orthoreovirus Mamífero 3/efectos de la radiación , Rayos Ultravioleta , Regulación hacia ArribaRESUMEN
Based on the known capacity of deoxynivalenol (DON) to target gut lymphoid tissue and IgA production, it was hypothesized that this mycotoxin interferes with the immune response to enteric reovirus infection. When mice were orally gavaged, first with 25 mg/kg bw DON, and then with reovirus serotype 1, strain Lang (T1/L) 2 or 12 h later, viral titers in the GI tract were 10-fold higher than control mice after 5 days. Virus was almost completely cleared in both treatment and control groups from intestinal tissue after 10 days. Real-time PCR indicated that, in infected control mice, reovirus lambda2 core spike (L2 gene) RNA per g feces in infected mice that were pretreated with DON was significantly higher at 1, 3, and 5 days than in infected mice only. In reovirus-infected mice, DON at doses of 10 and 25 mg/kg bw but not 2 and 5 mg/kg bw increased fecal L2 RNA, whereas DON doses as low as 2 mg/kg potentiated L2 RNA levels in Peyer's patches (PP). Reovirus-specific IgA levels in feces of mice treated with DON were significantly elevated, as were specific IgA responses in lamina propria and PP fragment cultures. Similar effects were observed for serum IgA and IgG. DON suppressed IFN-gamma responses in PP to reovirus at 3 and 5 days as compared to infected controls, while IL-2 mRNA concentrations were unaffected. Although reovirus alone did not induce Th2 cytokine mRNAs in PP, DON exposure significantly elevated IL-4, IL-6, and IL-10 mRNA expression at various times during the infection. ELISPOT revealed that mRNA expression data corresponded to suppression of IFN-gamma- and enhancement of IL-4-producing cell responses in PP cultures from DON-treated mice. Taken together, these data suggest that DON transiently increased both severity of the reovirus infection and shedding in feces as well as elevated reovirus IgA responses. These effects corresponded to suppressed Th1 and enhanced Th2 cytokine expression.
Asunto(s)
Infecciones por Reoviridae/inmunología , Tricotecenos/toxicidad , Animales , Anticuerpos Antivirales/biosíntesis , Relación Dosis-Respuesta a Droga , Heces/virología , Femenino , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Interferón gamma/biosíntesis , Intestinos/virología , Ratones , ARN Mensajero/análisis , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células Th2/efectos de los fármacos , Células Th2/inmunología , Factor de Crecimiento Transformador beta/genética , Esparcimiento de Virus/efectos de los fármacosRESUMEN
Systemic immunity is progressively impaired in aging, predisposing to morbidity and mortality from neoplasia and infectious disease. However, the effect of aging on mucosal immunity is controversial. To assess intestinal immunity in aging, young and aged mice were orally exposed to reovirus or cholera toxin (CT) and specific antibody and reovirus-specific cytotoxic T-cell (CTL) responses were assessed. As previously reported, aged mice immunized orally with CT mounted diminished intestinal IgA responses to CT compared to young mice. In contrast, aged mice yielded two to three-fold more reovirus-specific IgA-producing cells in the Peyers's patches (PP) compared to young mice, and higher titers of reovirus-specific IgA in fragment culture supernatants. Cytotoxicity and CTL frequencies from aged mice were not different from those of young mice. Together, these results suggest a diminished potential for systemic and intestinal immunity to orally applied protein antigens in aging, but an intact ability to respond to intestinal virus infection. Infection with a replicating virus may induce inflammatory mediators and innate immune factors that potentiate the priming of mucosal immunity; overcoming aging related deficits otherwise observed following oral immunization with non-replicating antigens, and suggests the importance of antigen replication to antigen-specific immunotherapy strategies in the elderly.
Asunto(s)
Envejecimiento/inmunología , Mucosa Intestinal/inmunología , Orthoreovirus de los Mamíferos/inmunología , Infecciones por Reoviridae/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Médula Ósea/inmunología , Citotoxicidad Inmunológica , Inmunidad Mucosa , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Bazo/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Mucosal and parenteral immunizations elicit qualitatively distinct immune responses, and there is evidence that mucosal immunization can skew the balance of T helper 1 and T helper 2 responses. However, a clear picture of the effect of the route of infection on the balance of the T helper responses has not yet emerged. Our laboratory previously demonstrated that oral reovirus infection elicits specific serum immunoglobulin G2a (IgG2a), while parenteral reovirus infection elicits the mixed production of specific serum IgG2a and IgG1 in mice of the H-2(d) haplotype. Knowing that IgG2a production is indicative of a T helper 1 response and IgG1 production is indicative of a T helper 2 response, we hypothesized that the route of infection influences the development of T helper 1 and T helper 2 responses. Using quantitative reverse transcription-PCR, we found that mRNA for the T helper 1 cytokines gamma interferon and interleukin-12 (IL-12) were expressed in draining lymphoid tissues following both oral and parenteral infections. However, we observed that mRNA for the T helper 2 cytokine IL-10 was suppressed in the Peyer's patches and mesenteric lymph nodes and IL-4 mRNA was suppressed in the mesenteric lymph nodes compared to noninfected controls, following oral infection. Using recombinant cytokines and cytokine knockout mice, we confirmed that IL-4 plays a major role in mediating the route-of-infection-dependent differences in serum IgG subclass responses. Therefore, the route of infection needs to be taken into consideration when developing vaccines and adjuvant therapies.
Asunto(s)
Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Interleucina-10/inmunología , Interleucina-4/inmunología , Membrana Mucosa/inmunología , Membrana Mucosa/virología , Infecciones por Reoviridae/inmunología , Administración Oral , Animales , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/inmunología , Inmunoglobulina G/biosíntesis , Interferón gamma/genética , Interleucina-10/genética , Interleucina-10/farmacología , Interleucina-12/genética , Interleucina-4/genética , Masculino , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina/antagonistas & inhibidores , Receptores de Interleucina/metabolismo , Receptores de Interleucina-10 , Infecciones por Reoviridae/virología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
It is well established that the route of infection affects the nature of the adaptive immune response. However, little is known about the effects of the route of exposure on development of cytotoxic T-lymphocyte (CTL) responses. Alternative antigen-presenting cell populations, tissue-restricted expression of class I major histocompatibility complex-encoded molecules, and unique T-cell receptor (TCR)-bearing cells in mucosal tissues could influence the selection and expansion of responder T cells. This study addresses the question of whether the route of virus infection affects the selection and expansion of subpopulations of virus-specific CTLs. Mice were infected orally or in the hind footpads with reovirus, and the repertoires of TCR beta-chains expressed on virus-specific CD8(+) T cells in Peyer's patches or lymph nodes and spleens were examined. CD8(+) cells expressing the variable gene segment of the TCR beta-chain 6 (Vbeta6) expanded in the spleens of mice infected by either route and in CTL lines established from the spleens and draining lymphoid tissues. Adoptively transferred Vbeta6(+) CD8(+) T cells from orally or parenterally infected donors expanded in reovirus-infected severe combined immunodeficient recipient mice and mediated cytotoxicity ex vivo. Furthermore, recovered Vbeta6(+) cells were enriched for clones utilizing uniform complementarity-determining region 3 (CDR3) lengths. However, sequencing of CDR3beta regions from Vbeta6(+) CD8(+) cells indicated that Jbeta gene segment usage is significantly more restricted in CTLs from orally infected mice, suggesting that the route of infection affects selection and/or subsequent expansion of virus-specific CTLs.