For qPCR analysis, total DNA was isolated from 1 g of faecal

For qPCR analysis, total DNA was isolated from 1 g of faecal selleck bio material according to Apajalahti et al[31], which included removing the undigested particles from the faecal material by three rounds of low-speed (200 �� g) centrifugation and collection of the bacterial cells with high-speed centrifugation (30 000 �� g) at 15��C for 15 min using a Beckman AvantiTM centrifuge (Fullerton, CA, USA) with the rotor JA 25.50 or JLA 16.250 rotor, respectively. The bacterial cells were lysed after centrifugation with a combination of freeze-thaw cycles (freezing for 1 h at -70��C and thawing for 15 min in a 37��C water bath), lysozyme and vortexing with glass beads. DNA concentrations were determined with a NanoDrop ND-1000 Spectrophotometer (NanoDrop products, Wilmington, DE, USA).

Design of qPCR assays Divergences detected by comparing the sequence data of 16S rRNA gene clone libraries of healthy controls and symptomatically sub-grouped IBS patients (diarrhoea-predominant IBS, IBS-D; constipation-predominant IBS, IBS-C; and mixed symptom-subtype IBS, IBS-M) were used as the basis for selection of qPCR targets[11]. Prior to cloning and sequencing, the faecal microbial genomes had been profiled and fractioned on the basis of genomic guanine-plus-cytosine content[11]. Partial 16S rRNA gene sequences encompassing the variable regions V1 and V2 combined from all four sample types were aligned using either the version Beta 2003-08-22 of ARB[11,32] or ClustalW 1.83[33]. For the ARB alignment, an aligned sequence database (ssu_jan04_corr_opt.arb) was downloaded from the ARB home page (http://www. and the in-house sequences[11] were aligned using the ARB-EDIT FastAlign function, followed by manual correction of the alignments with special attention to the ends of the sequences. Finally, the sequences were imported into an existing tree file of the database (Tree-Bacteria) by filtering the data against a sequence of similar length as the imported partial 16S rRNA Drug_discovery gene sequences. Regions of the tree where sequences derived from one subject group (healthy vs IBS or healthy vs IBS subtypes) dominated over the other groups were considered as potentially interesting. In addition, a ClustalW 1.83 alignment (FAST DNA pairwise alignment algorithm option, gap penalty 3, word size 4, number of top diagonals 1 and window size 1) was constructed covering approximately 450 bp from the 5�� end of the 16S rRNA gene and visually inspected and cut from the Escherichia coli position 430 (universally conserved GTAAA) with BioEdit version[34]. Distance matrices were calculated from the ClustalW alignment with Phylip 3.66 Dnadist[35] using Jukes-Cantor correction.

Moreover, western-blot analyses revealed expression of MMP13 prot

Moreover, western-blot analyses revealed expression of MMP13 protein in the group administered HA/PEI/pMMP13, but not in other groups (Figure 7e). Figure 7 Expression of MMP13 proteins in liver tissues of fibrotic mice. Expression of EGFP and MMP13 proteins in liver tissues was visualized by fluorescent microscopy and immunoblotting, respectively. (a,c) Representative phase-contrast and selleck products (b,d) fluorescence … Figure 8 Hepatic collagen deposition and serum AST after pMMP13 gene therapy. Collagen deposition was tested for the (a) liver tissues from normal mice, (b) fibrotic mice treated with saline, (c) HA/PEI/pVector, or (d) HA/PEI/pMMP13. Bar = 400 mm. The … Antifibrotic effect of pMMP13 administered in HA/PEI complexes Intravenous administration of pMMP13 in HA/PEI complexes ameliorated liver fibrosis in mice.

Sirius red staining of liver sections showed the presence of collagen deposition in the liver tissues of CCl4-treated mice administered saline (Figure 8b). Collagen was deposited to a similar extent in liver tissues of CCl4-treated mice administered pVector in HA/PEI complexes (Figure 8c). In contrast, injections of pMMP13/HA/PEI complexes significantly decreased the deposition of collagen in CCl4-treated mice (Figure 8d), reducing collagen to levels similar to those in untreated normal mouse livers (Figure 8a). The administration of pMMP13 in HA/PEI complexes also promoted recovery from liver damage in the liver fibrosis model, significantly decreasing the plasma levels of aspartate aminotransferase (AST) compared with those in animals administered pVector in HA/PEI complexes.

Consistent with the collagen-deposition histology data, there was no significant difference in serum AST levels between the untreated normal group and the liver fibrosis group treated with pMMP13 in HA/PEI complexes (Figure 8e). Discussion In this study, we demonstrated the antifibrotic effects of pMMP13 administered in HA-shielded complexes in a murine liver fibrosis model. Treatment of mice with liver fibrosis by administering HA/PEI/pMMP13 increased MMP13 mRNA and protein levels in liver tissue. Moreover, HA/PEI/pMMP13 treatment reduced collagen I deposition in liver tissue and restored plasma AST levels to values similar to those in untreated normal mice. The shielding of PEI/pMMP13 with HA provided protection against PEI cytotoxicity.

The mechanism underlying this cytoprotective effect remains to be elucidated. However, Moghimi et al.23 reported that PEI cytotoxicity was associated with activation of caspase-2 and changes in the mitochondrial GSK-3 membrane potential. Thus, it is possible that neutralization of the cationic charges of PEI by HA reduces the mitochondrial damage caused by PEI. Moreover, in vivo, the shielding of PEI/pMMP13 with HA may prevent the interaction of PEI with plasma proteins of the circulatory system.

21) was prepared from pooled plasma of healthy donors and labeled

21) was prepared from pooled plasma of healthy donors and labeled with 3H-cholesteryl ester (PerkinElmer) essentially as described (13), and 1 million dpm of the 3H- cholesteryl ester-HDL were injected via the tail vein into mice that had been given P-407 i.p. 30 min before as detailed above. Blood was drawn 4 h later by heart puncture, TNF-�� inhibitor VLDL was isolated from 200 ��l of plasma as described above, and counts within VLDL were determined by scintillation counting (Beckman LS6500, Palo Alto, CA). Analysis of nascent VLDL composition VLDL (d < 1.006) was isolated from 200 ��l of plasma from each mouse obtained 180 min after P-407 injection as described (14). Enzymatic methods were used to quantitate total cholesterol, free cholesterol, triglycerides, and phospholipids as detailed above.

The bicinchoninic acid method (Pierce, Rockford, IL) was used to quantitate protein. MTP activity assay Assessment of microsomal triglyceride transfer protein (MTP) activity in mouse livers was performed using a fluorigenic assay essentially as described (15, 16). Briefly, to prepare microsomes, mouse liver pieces were washed with PBS, homogenized in 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, 250 mM sucrose, and 0.02% sodium azide using a Polytron homogenizer, and centrifuged (10,900 rpm, 30 min, 4��C; Beckman microcentrifuge). Supernatants were adjusted to pH 5.1 with concentrated HCl, mixed in the cold for 30 min, and centrifuged (13,000 rpm, 30 min, 4��C; Beckman microcentrifuge). Pellets were resuspended in 1 mM Tris-Cl, pH 7.6, 1 mM EGTA, and 1 mM MgCl2, vortexed, incubated for 30 min at 4��C, and ultracentrifuged (50,000 rpm, 4��C, 1 h; SW55 Ti rotor).

Supernatants were used for protein determination and MTP activity measurements in a reaction mixture (100 ��l) containing 3 ��l of donor vesicles [1.2 nmol of phosphatidylcholine (PC) and 100 pmol of fluorescent lipids], 3 ��l of acceptor vesicles (7.2 nmol of PC) in 10 mM Tris, pH 7.4, 0.1% BSA, and 150 mM NaCl buffer. To determine the percentage of lipid transfer, fluorescence values obtained from control assays containing no MTP source (blanks) were subtracted from sample values and then divided by the total fluorescence present in the vesicles reduced by blanks. Analysis of gene expression by real-time quantitative PCR Total RNA from mouse livers was isolated using trizol (Invitrogen), and quantified with a NanoDrop ND-100 UV-Vis spectrophotometer.

cDNA synthesis was performed from 1 AV-951 ��g of total RNA using reagents from Applied Biosystems (Darmstadt, Germany). Real-time quantitative PCR was carried out on an ABI-Prism 7700 (Applied Biosystems) sequence detector with the default settings. PCR primers and fluorogenic probes were designed with the Primer Express Software (Applied Biosystems) and synthesized by Eurogentec (Seraing, Belgium).

Probes were

Probes were labeled using 50 ��Ci [��-32P]ATP (MP Biomedicals, Solon, OH) and then purified on Quick Spin Sephadex G25 columns (Roche). Sequences for miRNA378, 378*, 199b, 103, and 21 probes are respectively the following: 5��-ggccttctgactccaagtccag-3��, 5��-acacaggacctggagtcaggag-3��, 5��-gaacagatagtctaaacactggg-3��, 5��-tcatagccctgtacaatgctgct-3��, and 5��-tcaacatcagtctgataagcta-3��. Membrane was prehybridized in rapid hybridization buffer (GE Healthcare Life Sciences, Piscataway, NJ) at 42��C. After denaturing the probe, the membrane was incubated overnight at 42��C in hybridization buffer and then washed three times for 7 min at 32��C in SSC 2��, SDS 0.1%. Quantification of Northern blots was performed by phosphorimaging using Quantity One Software (Bio-Rad). Triacylglycerol measurements.

The amount of cellular triacylglycerol was determined with Infinity triglyceride reagent (Sigma-Aldrich, St. Louis, MO) with glycerol as the standard. RESULTS Ago2 is required for efficient synthesis of triacylglycerols. Ago2 plays a key role in the processing and stability of miRNAs, as well as action of endogenous siRNAs; therefore, to investigate potential roles of miRNAs on aspects of adipocyte biology, we evaluated effects of Ago2 deficiency on adipocyte differentiation and metabolism (3). We stably knocked down expression of Ago2 in 3T3-L1 preadipocytes using a retroviral shRNA expression vector (shAgo2) (20). Ago2 mRNA was reduced by ~72% in Ago2 knock-down adipocytes at day 7 of differentiation (Fig. 1A). By phase-contrast microscopy, the rate and extent of adipogenesis did not appear to be different.

Similarly, adipocyte morphology including size and number of lipid droplets appear similar (Fig. 1B). To confirm and extend these observations, we quantified expression of several adipocyte genes and found that C/EBP�� mRNA levels are similar between control and shAgo2 3T3-L1 preadipocytes and adipocytes at day 8 (Fig. 1C). However, Western blot analysis revealed a slight but significant increase in expression of PPAR�� and C/EBP�� proteins in adipocytes with an Ago2 deficiency, although differences in FABP4 were not observed (Fig. 1D). Taken together, these data suggest that differentiation of adipocytes is not substantially influenced by knockdown of Ago2. Fig. 1. Ago2 is required for efficient incorporation of [14C]glucose into triacylglycerol.

A: knockdown of Ago2 in 3T3-L1 adipocytes. After retroviral transduction with shRNAs, Ago2 expression levels in day 7 3T3-L1 adipocytes were measured by quantitative RT-PCR, … To assess a potential Cilengitide role of Ago2 in lipid metabolism, we examined effects of Ago2 knock-down on lipogenesis. After metabolic labeling with [14C]acetate for 45 min or with [14C]glucose for 2 h, lipids were extracted and separated by thin-layer chromatography (Fig. 1, E and F, respectively).

TMA block sections were subjected to an immunohistochemical analy

TMA block sections were subjected to an immunohistochemical analysis selleck chem inhibitor using anti-CRKL monoclonal antibody (Y243; 1:100 dilution), Histofine Simple Stain Max-Po (Multi), and 3,3′-diaminobenzidine … DNA fluorescence in situ hybridization (FISH) FISH was performed as previously described [16-19]. Tissue slides were hybridized with a Spectrum Orange-labeled BAC clone (RP11-801O20 and RP11-1058B20) for the CRKL locus (Advanced GenoTechs Co., Tsukuba, Japan) and a Spectrum Green-labeled control probe for the near centromere locus on chromosome 22 (BAC clone: RP11-232E17). 4′,6-Diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) was used for nuclear staining (Figure (Figure33). MTT assay and direct cell counting In the experiment involving treatment with the CRKL-targeting peptide, an MTT assay was performed to assess cell viability in Figure Figure4G.

4G. The cells were cultured with the indicated concentration of CRKL-targeting peptide or dimethyl sulfoxide (DMSO) at 37��C for 72h, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO) was then added at a final concentration of 0.25mg/mL. After incubation at 37��C for 4h, absorbance was measured at a wavelength of 570nm using a microplate reader. Cells grown in complete medium with DMSO alone were used as controls. The final concentration of DMSO was set to 0.2%. To assess cell proliferation in Figure Figure4H,4H, the cells were cultured with CRKL targeting peptide or DMSO at 37��C for 72h. Cell proliferation was measured by directly counting the cells using a hemocytometer, as described previously [20].

Figure 4 Responses of the MKN74 gastric cancer cell line withCRKLamplification to treatment with BMS354825 (a dual Src/BCR-ABL kinase inhibitor) and CRKL-targeting peptide. (A) Viability of MKN74 cells treated with BMS354825 but not those treated with AMN107 (a … QRT-PCR Total RNA was extracted using Isogen (Nippongene, Tokyo, Japan) or an RNeasy Plus mini kit (Qiagen, Valencia, CA) and converted to cDNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Real-time QRT-PCR was performed using the cDNA and Fast SYBR Green Master Mix (Applied Biosystems, Foster City, CA) on a StepOne Real-Time PCR system (Applied Biosystems).

The following PCR primers were used: 5′-CAA CCT GCC TAC AGC AGA AGA TAA-3′ and 5′-CGG CAT CAT TCC CAG GAA-3′ for the CRKL transcript, Entinostat and 5′-GGT GGT CTC CTC TGA CTT CAA CA-3′ and 5′-GTT GCT GTA GCC AAA TTC GTT GT-3′ for the transcript of a housekeeping gene, GAPDH. The relative amounts of CRKL transcript were normalized to those of the GAPDH transcript. Western blot analysis Cells were lysed, and the protein concentration was quantified using a BCA protein assay kit (Pierce, Rockford, IL). The proteins were electrophoresed and transferred to a PVDF membrane (GE Healthcare Bio Science, Piscataway, NJ).

However, little is known about the situation for biliary tract ca

However, little is known about the situation for biliary tract cancer (BTC), unless a rare tumor with a grim prognosis and limited treatment options. Two recent studies showed expression of IGF-1R and its ligands in gallbladder carcinoma (GBC)[40] and cholangiocarcinoma (CC) specimens[41]. Therefore, the objectives of the current study were to investigate IGF-1R expression in BTC cell lines and to evaluate the efficacy of in vitro treatment with selective IGF-1R inhibitor NVP-AEW541 alone or in combination with gemcitabine, 5-fluorouracil (5-FU) or Polo-like kinase 1 inhibitor BI 2536, which is currently being investigated in phase II studies including our hospital for the treatment of solid tumors[42].

MATERIALS AND METHODS Drugs and cells Seven BTC cell lines; five extrahepatic CC cell lines (EGI-1, TFK-1, CC-SW-1, CC-LP-1, and SK-ChA-1)[43-47] and two GBC cell lines (Mz-ChA-1, Mz-ChA-2)[46], were examined. All cell lines were cultured in a 37��C incubator with 5%-10% CO2 in appropriate media, which were changed every 3 d. NVP-AEW541 (targeting IGF-1R) was obtained from Novartis (Basel, Switzerland), dissolved in dimethyl sulfoxide (DMSO) (as 10 mmol/L stock) and stored at -20��C according to manufacturer��s instructions. BI 2536 (targeting Plk-1) was kindly provided by Boehringer (Ingelheim, Germany). Gemcitabine and 5-FU (diluted in 0.9% NaCl) were provided by our hospital pharmacy. Inhibition of cell growth Cytotoxic effects of drugs alone and in combination were determined by automated cell counting (Casy Cell Counter Model TT; Innovatis AG, Reutlingen, Germany) according to manufacturer��s instructions.

Briefly, 2 �� 105 cells were seeded in duplicates in T25 flasks with media containing the designated drugs or vehicle control followed by incubation for 3 or 6 d. For the 6 d experiment, medium was changed after 3 d and treatment repeated. At the end of incubation, cells were trypsinized, washed, and analyzed in triplicates by automated cell counting. Immunoblotting Cell culture monolayers were washed with ice-cold PBS and lysed in flask with a buffer containing Tris-HCl (50 mmol/L, pH 7.4), NP-40 (10 g/L), NaCl (200 mmol/L), sodium-orthovanadate (200 mmol/L), 2-glycerophosphate (1 mmol/L), sodium fluoride (20 mmol/L), DTT (10 mmol/L), PMSF (200 mmol/L) and 0.2% proteinase inhibitor cocktail (Sigma-Aldrich, Munich, Germany) on ice for 30 min. The lysate was then centrifuged at 13 000 r/min for 15 min and proteins in supernatant were quantified by Bradford protein assay (Bio-Rad, Munich, Germany) and stored AV-951 at -80��C.


AUTHORS�� DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a ��U�� Palbociclib manufacturer are those for which no compensation was received; those relationships marked with a ��C�� were compensated. For a detailed description of the disclosure categories, or for more information about ASCO’s conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Michael C. Heinrich, Novartis (C), MolecularMD (U), Pfizer Inc (C); Christopher L.

Corless, Novartis Inc (U); Christopher W. Ryan, Novartis Inc (C), Pfizer Inc (C); Margaret von Mehren, Novartis Inc (C); Robert S. Benjamin, Novartis Inc (C); George D. Demetri, Novartis Inc (C), Pfizer Inc (C), Infinity Pharmaceuticals (C) Stock Ownership: Michael C. Heinrich, MolecularMD Honoraria: Michael C. Heinrich, Novartis Inc, Pfizer Inc; Christopher L. Corless, Novartis Inc; Christopher W. Ryan, Novartis Inc, Pfizer Inc; Margaret von Mehren, Novartis Inc; Robert S. Benjamin, Novartis Inc; George D. Demetri, Novartis Inc, Pfizer Inc Research Funding: Michael C. Heinrich, Novartis Inc, Pfizer Inc; Christopher L. Corless, Novartis Inc; Christopher W. Ryan, Novartis Inc, Pfizer Inc; Margaret von Mehren, Novartis Inc; Charles D. Blanke, Novartis Inc; Robert S.

Benjamin, Novartis Inc; George D. Demetri, Novartis Inc, Pfizer Inc, Infinity Pharmaceuticals Expert Testimony: George D. Demetri, Novartis Inc (U), Pfizer Inc (U), Infinity Pharmaceuticals (U) Other Remuneration: Robert S. Benjamin, Novartis Inc AUTHOR CONTRIBUTIONS Conception and design: Michael C. Heinrich, Christopher L. Corless, Ernest C. Borden, Christopher D.M. Fletcher, Christopher W. Ryan, Margaret von Mehren, Charles D. Blanke, Cathryn Rankin, Robert S. Benjamin, Vivien H. Bramwell, George D. Demetri, Monica M. Bertagnolli, Jonathan A. Fletcher Financial support: Michael C. Heinrich, Kouros Owzar, Christopher L. Corless, Ernest C. Borden, Cathryn Rankin, Monica M. Bertagnolli Administrative support: Michael C. Heinrich, Donna Hollis, Ernest C. Borden, Christopher W. Ryan, Margaret von Mehren, Charles D. Blanke, Cathryn Rankin, Robert S. Benjamin, George D. Demetri, Monica M. Bertagnolli, Jonathan A. Fletcher Provision of study materials or patients: Michael C. Heinrich, Christopher L. Corless, Ernest C. Borden, Christopher D.M. Fletcher, Christopher W. Ryan, Margaret Batimastat von Mehren, Charles D. Blanke, Robert S.

, 2010) However, a further possibility is that the role of SDP o

, 2010). However, a further possibility is that the role of SDP on adolescent smoking trajectories selleck chemicals Abiraterone may operate through the mother��s smoking behavior in the child��s adolescence. Mothers who SDP are very likely to continue smoking through the child��s early life and adolescence (Floyd et al., 1993). Studies suggest that this type of exposure to parental smoking may solidify a child��s values and beliefs about smoking and demonstrate prosmoking norms (for review see Chassin, Presson, Rose, & Sherman, 1998; Darling & Cumsille, 2003). Although there is debate about the relative strength of parents�� influence on their children��s smoking behaviors (Chassin, Presson, Sherman, Montello, & McGrew, 1986), studies have generally observed that mothers (as opposed to fathers) have the strongest influence during adolescence (Avenevoli & Merikangas, 2003).

The cognitive and developmental problems associated with SDP briefly reviewed above, combined with the large body of research on cognitive and developmental origins of youth problem behaviors (see Smith, Leve, & Chamberlain, 2011) substantiate how the effects of SDP might be mediated by child and youth behavior problems. Only two studies, to our knowledge, have explored whether the effect of SDP operates through childhood behavior problems to influence tobacco use during adolescence (Cornelius et al., 2005; Griesler et al., 1998). Both studies observe a positive relationship between SDP and regular smoking among adolescents that is mediated by childhood behavior problems.

In this preliminary research, the findings have held after controlling for SES and concurrent smoking by the mother (Griesler et al., 1998). It is not clear whether these findings will also hold in larger datasets with data collected over longer time periods, which allows more extensive analyses of confounders and potential methodological bias. Further research could also illuminate how SDP might influence the trajectories and progression of youth smoking. In the research presented in this paper, we extend the work of Weden and Miles (2011) to include a test of the mediation through problem behavior hypothesis. In their work, Weden and Miles used multinomial logistic regression to examine whether patterns of maternal smoking (before, during, and after pregnancy) were associated with smoking patterns in their offspring, finding that maternal smoking during pregnancy was associated Brefeldin_A with an increased likelihood of youth smoking. In this paper, we examine the potential for measures of problem behavior to mediate those relationships.

We used two different measures of price The first measure is a s

We used two different measures of price. The first measure is a self-reported price for the most recent purchase selleck chem of a pack of 20 cigarettes. To address its potential endogeneity, we created a second price measure, which is an average state/province cigarette price. We calculated this by averaging the self-reported price of the most popular country brand across state/province observations. Both price measures were converted into international dollars using the purchasing power parity. An extended discussion and justification of the price measures can be found in Ross, Blecher, Yan, and Hyland (in press). Using the second price measure, we estimated the average price of the most popular brand in the last quarter of 2002 in the United States ($3.33 for Marlboro), Australia ($4.05 for Winfield), Canada ($4.

43 for Du Maurier), and the United Kingdom ($5.87 for Lambert and Butler). In addition to these two price measures, we also tested alternative exogenous price that was available in the United States and Canada only. In the United States, we used prices from AC Nielsen and from Orzechowski and Walker (2008), in Canada, we obtained province level prices from the Statistics Canada. The AC Nielsen prices reflect price promotions/discounts and were consistently lower than the self-reported prices from the survey. The prices in Orzechowski and Walker represent full retail prices and were consistently higher than the self-reported prices. For instance in the United States, the average AC Nielsen price was $3.02 and the average price from Orzechowski and Walker was $3.60.

There are three dichotomous indicators for the level of education/income, low, medium, and high to capture the differences across the four countries between their different education systems and income distribution. Brefeldin_A See (Thompson et al. (2006) for the details. We controlled for nicotine dependence by the number of cigarettes consumed per week. The extent of price promotions is measured by a cross country average of five dichotomous indicators for sales promotion activities: cigarette advertisements/promotions on shop/store windows or inside shops/stores, free samples of cigarettes, cigarette price offers, free gifts or special discount offers, and competitions linked to cigarettes. The U.S. smokers were most exposed to these promotions with the average smoker noticing 2.69 activities in the last six months before the survey. United Kingdom, Australian, and Canadian smokers were exposed to 1.75, 1.16 and 1.01 promotion activities, respectively. This variable is used for descriptive purposes only since the country dichotomous indicators capture the different level of the price promotion exposure in the models.

Following a 3-week protein treatment (intravenous, 300 ��g/head),

Following a 3-week protein treatment (intravenous, 300 ��g/head), total RNA was extracted from isolated tu
Patients with cystic fibrosis (CF), an autosomal recessively inherited disease caused by a mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, are particularly susceptible Temsirolimus chemical structure to pulmonary infections with Pseudomonas aeruginosa [1,2]. Colonization of the airways of CF patients with P. aeruginosa results in higher morbidity and mortality because of the faster decline of the lung function, especially from the chronic infection phase onwards [3-5]. Detection of colonization and infection by this pathogen as early as possible enables to postpone the chronic infective stage and eventually to achieve the eradication of P. aeruginosa through early treatment.

Indeed, early aggressive antibiotic therapy is now generally accepted as an efficient means to postpone chronic colonization [6,7]. In most routine laboratories detection of bacterial species in respiratory samples is achieved by culture. However, it has been shown that routine culture of sputa from CF patients yields limited microbiological information since it frequently fails to identify the pathogens, which were shown to be present by means of PCR [8]. Furthermore, the correct detection and identification of P. aeruginosa, although in general not a fastidious organism, is not as straightforward as frequently assumed [9,10].

To circumvent culture associated limitations, several molecular assays for the detection of Pseudomonas species have been described [8,11-19], D?ring and colleagues [20] correctly remarked that, because of the influence of sample pretreatment, DNA-extraction protocol and the PCR format, there is a need for validation of the PCR techniques before these can be used in a routine laboratory. However, to our knowledge, no study systematically compared the sensitivity of different culture, DNA-extraction, PCR and real-time PCR methods for the detection of P. aeruginosa from CF sputum, by using a CF patient sputum based dilution series of P. aeruginosa. Here, we compared the sensitivity of three culture media, five DNA-extraction protocols, two conventional PCR formats and four real-time PCR formats for the detection of P. aeruginosa, using a dilution series of P. aeruginosa positive sputa in a pool of P. aeruginosa negative sputa.

Results In this study, we compared the sensitivity of different culture and PCR methods. To that purpose, we prepared a P. aeruginosa dilution series in CF sputum by diluting P. aeruginosa positive CF patient sputa in a pool of P. aeruginosa negative CF patient sputa. This was done instead of diluting cultured P. aeruginosa cells in saline or diluting P. aeruginosa positive sputum in saline or spiking sputa with P. aeruginosa cells, to mimick as closely as possible the sputum samples sent Carfilzomib to routine laboratories.