Monomeric calgranulins measured by SELDI-TOF mass spectrometry and calprotectin measured by ELISA as biomarkers in arthritis

BACKGROUND: SELDI-TOF mass spectrometry (MS) is a high-throughput proteomic approach with potential for identifying novel forms of serum biomarkers of arthritis. METHODS: We used SELDI-TOF MS to analyze serum samples from patients with various forms of inflammatory arthritis. Several protein profiles were collected on different Bio-Rad Laboratories ProteinChip arrays (CM10 and IMAC-Cu(2+)) and were evaluated statistically to select potential biomarkers. RESULTS: SELDI-TOF MS analyses identified several calgranulin proteins [S100A8 (calgranulin A), S100A9 (calgranulin B), S100A9*, and S100A12 (calgranulin C)], serum amyloid A (SAA), SAA des-Arg (SAA-R), and SAA des-Arg/des-Ser (SAA-RS) as biomarkers and confirmed the results with other techniques, such as western blotting, immunoprecipitation, and nano-LC-MS/MS. The S100 proteins were all able to significantly differentiate samples from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from those of patients with inflammatory bowel diseases used as an inflammatory control (IC) group, whereas the SAA, SAA-R, and SAA-RS proteins were not, with the exception of AS. The 4 S100 proteins were coproduced in all of the pathologies and were significantly correlated with the plasma calprotectin concentration; however, these S100 proteins were correlated with the SAA peak intensities only in the RA and IC patient groups. In RA, these S100 proteins (except for S100A12) were significantly correlated with the serum concentrations of C-reactive protein, matrix metalloproteinase 3, and anti-cyclic citrullinated peptide and with the Disease Activity Score (DAS(28)). CONCLUSIONS: The SELDI-TOF MS technology is a powerful approach for analyzing the status of monomeric, truncated, or posttranslationally modified forms of arthritis biomarkers, such as the S100A8, S100A9, S100A12, and SAA proteins. The fact that the SELDI-TOF MS data were correlated with results obtained with the classic calprotectin ELISA test supports the reliability of this new proteomic technique.     

Differences in biofilm development and antibiotic susceptibility among Streptococcus pneumoniae isolates from cystic fibrosis samples and blood cultures

OBJECTIVES: To compare the capability of biofilm development between Streptococcus pneumoniae isolates from cystic fibrosis (CF) respiratory samples and those from non-CF blood cultures. Antibiotic susceptibility of biofilm-forming isolates, as well as differences between antibiotic susceptibility of sessile cells [minimum biofilm inhibitory concentration (MBIC)] and their planktonic counterparts (conventional MIC), were also assessed. METHODS: Biofilm formation was performed using a microtitre method in 20 CF and 22 non-CF blood culture S. pneumoniae isolates. RESULTS AND CONCLUSIONS: Biofilm formation occurs more frequently among S. pneumoniae isolates from CF (80%) than among non-CF blood culture isolates (50%) (P = 0.04). Moreover MBICs were significantly higher than conventional planktonic MICs among CF but not among non-CF blood isolates, suggesting a high adaptability of CF strains to form biofilms in adverse conditions.     

Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients

The conducting airways are the primary target for gene transfer in cystic fibrosis (CF), yet the inflammation associated with CF lung disease could potentially pose a significant barrier to gene transfer vectors, such as recombinant adeno-associated virus (rAAV). In order to investigate this possibility, aliquots of bronchoalveolar lavage (BAL) fluid from eight individuals with CF were tested for their in vitro inhibitory effects on rAAV transduction, along with BAL from non-CF individuals. While the non-CF BAL fluid was not inhibitory, seven of eight CF BAL samples had significant inhibitory activity, resulting in a five- to 20-fold reduction in transduction events. Inhibition of rAAV transduction by CF BAL could be reversed by alpha-1-antitrypsin (AAT), but not by DNase. When neutrophil elastase and neutrophil alpha defensins (human neutrophil peptides, HNP) were measured in these samples, they were elevated by 500- and 10,000-fold, respectively. The levels of HNP correlated inversely with the amount of rAAV transduction. Furthermore, rAAV transduction could be blocked by purified HNP in an AAT-reversible manner at HNP concentrations within the range measured in these fluids. We conclude that products of inflammation in CF BAL fluid are inhibitory to rAAV transduction, and that these effects may be reversible by AAT.     

Identification of serum amyloid A as a biomarker to distinguish prostate cancer patients with bone lesions

BACKGROUND: Prostate cancer has a propensity to metastasize to the bone. Currently, there are no curative treatments for this stage of the disease. Sensitive biomarkers that can be monitored in the blood to indicate the presence or development of bone metastases and/or response to therapies are lacking. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is an affinity-based approach that allows sensitive and high-throughput protein profiling and screening of biological samples. METHODS: We used SELDI-TOF MS for protein profiling of sera from prostate cancer patients (n = 38) with and without bone metastases in our effort to identify individual or multiple serum markers that may be of added benefit to those in current use. Serum was applied to ProteinChip surfaces (H4 and IMAC) to quickly screen samples and detect peaks predominating in the samples obtained from patients with bone metastases. Unique proteins in the bone metastasis cohort observed by SELDI-TOF MS were identified by two-dimensional gel electrophoresis, in-gel trypsin digestion, and tandem MS. The identities of the proteins were confirmed by ELISA and immunodepletion assays. RESULTS: The cluster of unique proteins in the sera of patients with bone metastases was identified as isoforms of serum amyloid A. Machine-learning algorithms were also used to identify patients with bone metastases with a sensitivity and specificity of 89.5%. CONCLUSIONS: SELDI-TOF MS protein profiling in combination with other proteomic approaches may provide diagnostic tools with potential clinical applications and serve as tools to aid in the discovery of biomarkers associated with various diseases.     

Calcium oxalate crystallization in untreated urine, centrifuged and filtered urine and ultrafiltered urine.

Centrifuged and filtered urine is often used to evaluate in vitro the crystallization processes of calcium oxalate (CaOx), but even such simple manipulations can alter the composition of the urine, as regards its protein and lipid concentrations. In urine samples taken from 17 normal male adults, we evaluated CaOx crystallization by simultaneously using three different types of urine: untreated (U), centrifuged at 2000 rpm (800 g) and filtered at 0.22 microm (CF), and centrifuged-filtered and ultrafiltered at 10 000 Da (CFU). The addition of 1.2 mmol/l of oxalate to each type of urine produced notably different results. The total amount of CaOx crystals (expressed as calcium oxalate dihydrate crystals (COD) + oxalate monohydrate crystals (COM) area/total area x 100) was on average 13.2% in U urine, 70.7% in CF urine and 11.1% in CFU urine (CF > U and CFU, U = CFU); the relative prevalence of COD and COM (expressed as COD area/COM area) was on average 71.4 in U urine, 0.0026 in CF urine and 5.5 in CFU urine (U > CF and CFU, CFU > CF); the diameter of COD (expressed in microns) was on average 15.2 in U urine, 3.7 in CF urine and 24.3 in CFU urine (CFU > U and CF, U > CF); the diameter of COM (expressed in microns) was on average 5.2 in U urine, 2.6 in CF urine and 8.9 in CFU urine (CFU > U and CF, U > CF); the total amount of CaOx aggregates (expressed as CaOxAgg area/total area x 100) was on average 8.5% in U urine, 22.1% in CF urine and 2.9% in CFU urine (CF > U and CFU, U > CF). We conclude that CaOx crystallization processes in manipulated urine are extremely different, probably due to changes in macromolecular compounds.     

The thymidine-dependent small-colony-variant phenotype is associated with hypermutability and antibiotic resistance in clinical Staphylococcus aureus isolates

Thymidine-dependent small-colony variants (TD-SCVs) of Staphylococcus aureus can be isolated from the airway secretions of patients suffering from cystic fibrosis (CF) and are implicated in persistent and treatment-resistant infections. These characteristics, as well as the variety of mutations in the thymidylate synthase-encoding thyA gene which are responsible for thymidine dependency, suggest that these morphological variants are hypermutable. To prove this hypothesis, we analyzed the mutator phenotype of different S. aureus phenotypes, in particular CF-derived TD-SCVs, CF-derived isolates with a normal phenotype (NCVs), and non-CF NCVs. The comparative analysis revealed that the CF isolates had significantly higher mutation rates than the non-CF isolates. The TD-SCVs, in turn, harbored significantly more strong hypermutators (mutation rate > or = 10(-7)) than the CF and non-CF NCVs. In addition, antimicrobial resistance to non-beta-lactam antibiotics, including gentamicin, ciprofloxacin, erythromycin, fosfomycin, and rifampin, was significantly more prevalent in TD-SCVs than in CF and non-CF NCVs. Interestingly, macrolide resistance, which is usually mediated by mobile genetic elements, was conferred in half of the macrolide-resistant TD-SCVs by the point mutation A2058G or A2058T in the genes encoding the 23S rRNA. Sequence analysis of mutS and mutL, which are involved in DNA mismatch repair in gram-positive bacteria, revealed that in hypermutable CF isolates and especially in TD-SCVs, mutL was often truncated due to frameshift mutations. In conclusion, these data provide direct evidence that TD-SCVs are hypermutators. This hypermutability apparently favors the acquisition of antibiotic resistance and facilitates bacterial adaptation during long-term persistence.     

The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways.

Cystic fibrosis (CF) airway cells, besides their well-known defect in cAMP-dependent Cl- conductance, are characterized by an enhanced Na+ conductance. In this study we have examined the Na+ conductance in human respiratory tract by measuring transepithelial voltage and resistance (Vte, Rte) and by assessing membrane voltages (Vm) of freshly isolated airway epithelial cells from CF and non-CF patients. Basal amiloride inhibitable (10 micromol/liter) equivalent short circuit current (Isc = Vte/Rte) was significantly increased in CF compared with non-CF tissues. After stimulation by forskolin (10 micromol/liter) a significant depolarization of Vm corresponding to the cAMP-dependent activation of a Cl- conductance was observed in non-CF but not in CF airway cells. In non-CF tissue but not in CF tissue the effects of amiloride and N-methyl-D-glucamine on Vm were attenuated in the presence of forskolin. Also the amiloride-inhibitable Isc was significantly reduced by forskolin (1 micromol/liter) and isobutylmethylxanthine (IBMX; 100 micromol/liter) only in non-CF tissue. We conclude that cystic fibrosis transmembrane conductance regulator acts as a downregulator of epithelial Na+ channels in human airways. This downregulation of epithelial Na+ channels is absent in CF airways, leading to hyperabsorption and to the characteristic increase in mucus viscosity.     

Genotypic analysis of respiratory mucous sulfation defects in cystic fibrosis.

Intracellular dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed to alter endosomal acidification. The most widely studied consequence of this defect has been alterations in the biochemical properties of cystic fibrosis (CF) respiratory mucus glycoproteins. However, studies confirming the existence of mucous processing defects in CF have been hindered by the lack of in vivo animal models by which to test these hypotheses in the absence of secondary effects of chronic bacterial infection. The human bronchial xenograft model has been useful in evaluating the pathophysiologic differences between CF and non-CF airway epithelium, in the absence of secondary disease effects such as goblet cell hyperplasia. In this study we sought to compare the extent of sulfation within secreted mucus glycoproteins from CF and non-CF human bronchial xenografts. Cumulative results of xenografts generated from 13 independent CF tissue samples demonstrated a statistically significant higher level of sulfation (1.7 +/- 0.18, P < 0.026) as compared to non-CF paired controls. Such findings add to the growing body of knowledge that primary defects in sulfation exist in CF respiratory mucin. Correlation of genotype with the extent of mucus sulfation revealed two categories of CF tissues with statistically different mucus sulfation profiles. Results from these studies demonstrated a 2.0 +/- 0.15-fold higher level of mucus sulfation produced from xenografts of five defined CF genotypes as compared to non-CF controls (P < 0.004, n= 10). Interestingly, three CF samples for which one mutant allele remained undefined (deltaoff8/unknown or G551D/unknown) demonstrated no statistical difference in the level of sulfation as compared with matched non-CF controls (n= 3). This as yet unknown allele was not identified within a screen for the 26 most common CF mutations. These results provide preliminary evidence for allelic variation within the CF population which may begin to elucidate the structure-function of CFTR with regards to intracellular mucus processing defects.     

Analysis of ligand binding by bioaffinity mass spectrometry

BACKGROUND: Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach. METHODS: BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS. RESULTS: A protein with 141kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at approximately 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with approximately 3kDa to approximately 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts. CONCLUSIONS: Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.     

Fluticasone propionate inhibits lipopolysaccharide-induced proinflammatory response in human cystic fibrosis airway grafts

Airway inflammation, one of the major factors leading to lung damage in cystic fibrosis (CF) patients, is associated with an abnormal increase in proinflammatory cytokines. In this work, we demonstrate the increased release of the proinflammatory cytokines after lipopolysaccharide (LPS) stimulation: human interleukin (hIL)-8 in CF and non-CF airway xenografts, and hIL-6 and human growth-related oncogene-alpha (hGRO-alpha), which could be only analyzed in non-CF xenografts. Under basal conditions, we observed that hIL-8 was higher in CF xenografts compared with non-CF. We also report the anti-inflammatory effect of a glucocorticoid, fluticasone propionate (FP), on CF airway epithelium using a humanized model of airway inflammation developed in nude mice. In CF and non-CF tracheal xenografts, airway inflammation was induced by inoculating Pseudomonas aeruginosa LPS (4 h; 1 microg/ml) in the lumen of the xenografts. FP pretreatment (2 h; 10(-8) M) followed by P. aeruginosa LPS stimulation induced a significant reduction of LPS-induced hIL-8 release in airway liquid collected from CF and non-CF tracheal xenografts (85 and 80%, respectively). In non-CF tracheal xenografts, FP treatment before LPS stimulation induced a significant decrease in hIL-6 and hGRO-alpha. From these data, we suggest that FP exerts anti-inflammatory properties that may be appropriate to CF therapy, at an early stage of the disease. In addition, these results demonstrate that the humanized airway model of inflammation provides a relevant tool for analyzing the effects of anti-inflammatory drugs in different diseases in which airway inflammation is implicated.     

Application of serum protein fingerprint in diagnosis of coronary artery disease

ObjectivesThe object of this study is to establish serum protein fingerprint models for early diagnosis and evaluation of luminal stenosis severity of CAD.Design and methodsA total of 189 samples were analyzed by surface-enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF MS) coupled with CM10 chips and bioinformatics tools.ResultsTen top-ranked proteins were selected as the potential fingerprints of pattern 1, which distinguishes patients with significant CAD (stenosis ≥ 50%) from control individuals. Blind test was analyzed on the second day with a specificity of 79.3% and sensitivity of 78.3%. Pattern 2 distinguishes significant CAD from minimal CAD with luminal stenosis between 25% and 50%. The specificity and sensitivity of blind test for pattern 2 are 77.3% and 73.9%, respectively.ConclusionsThe SELDI-TOF MS technique combined with bioinformatics approaches may be a novel noninvasive method which can not only facilitate the discovery of diagnostic models for CAD and its severity, but also provide a useful tool for molecular diagnosis.     

Pharmacokinetics of netilmicin in children with and without cystic fibrosis.

The pharmacokinetics of intravenous netilmicin was studied in cystic fibrosis (CF) and non-CF patients who were closely matched according to age. The serum concentrations showed a moderately higher variance within the CF group. The serum half-life in CF patients was 1.37 h compared with 2.29 h in the non-CF subjects (P less than 0.05). The apparent distribution coefficients were 0.306 and 0.356 liters/kg in the CF and non-CF groups, respectively. The mean body clearance was 6.6 liters/h per 1.73 m2 in the CF group compared with 5.3 liters/h per m2 in the non-CF controls, but the difference was not significant. The mean renal clearance in CF patients was 4.7 liters/h per 1.73 m2. From a pharmacokinetic point of view, the dosage of netilmicin required may be the same in CF as in non-CF patients.     

Role of the multidrug efflux system MexXY in the emergence of moderate resistance to aminoglycosides among Pseudomonas aeruginosa isolates from patients with cystic fibrosis

This study investigates the role of active efflux system MexXY in the emergence of aminoglycoside (AG) resistance among cystic fibrosis (CF) isolates of Pseudomonas aeruginosa. Three genotypically related susceptible and resistant (S/R) bacterial pairs and three other AG-resistant CF strains were compared to four non-CF strains moderately resistant to AGs. As demonstrated by immunoblot experiments, pump MexY was strongly overproduced in all of the resistant bacteria. This MexXY upregulation was associated with a 2- to 16-fold increase in the MICs of AGs in the S/R pairs and lower intracellular accumulation of dihydrostreptomycin. Alterations in mexZ, the repressor gene of operon mexXY, were found in all of the AG-resistant CF isolates and in one non-CF strain. Complementation of these bacteria with a plasmid-borne mexZ gene dramatically reduced the MICs of AGs, thus highlighting the role played by MexXY in the development of moderate resistance in CF patients. In contrast, complementation of the three non-CF strains showing wild-type mexZ genes left residual levels of resistance to AGs. These data indicate that a locus different from mexZ may be involved in overproduction of MexXY and that other nonenzymatic mechanisms contribute to AG resistance in P. aeruginosa.     

Effects of pretreatment protocols on human amniotic fluid protein profiling with SELDI-TOF MS using protein chips and magnetic beads

BackgroundThere is increasing interest in the use of human amniotic fluid (AF) proteomics with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for diagnosing pregnancy-associated abnormalities. A critical parameter of diagnostic biomarkers is the accuracy and reproducibility of protein patterns. We evaluated the effects of common pretreatment protocols on protein patterns generated using SELDI mass spectrometry with two different protein capture strategies (including functional protein chips and functionalized magnetic beads prior to MS analysis) in AF.MethodVarious extrinsic factors involved in processing and storing amniotic fluid, including matrix composition, sample storage time, temperature and freeze–thaw cycles, were analyzed regarding their impact on AF protein patterns using SELDI mass spectrometry with 2 different protein capture strategies.ResultsThree extrinsic factors (sample storage for 3 days at either room temperature or 4 °C or freeze–thawing the sample 5 times) significantly decreased the number or intensities of protein peaks detected in AF. Matrix dilutions also changed the spectra of AF, with more peaks and higher intensities observed with 50% α-cyano-4-hydroxycinnamic acid (CHCA). Moreover, protein chips captured more proteins or peptides than magnetic beads on SELDI-TOF MS profiling of AF.ConclusionsThese results suggest that extrinsic factors must be taken into account for valid data interpretation to ensure good reproducibility of AF profiling by SELDI mass spectrometry.     

Azithromycin selectively reduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells

Azithromycin (AZM) ameliorates lung function in cystic fibrosis (CF) patients. This macrolide has been suggested to have anti-inflammatory properties as well as other effects potentially relevant for therapy of CF. In this study, we utilized three CF (IB3-1, 16HBE14o- AS3, and 2CFSMEo-) and two isogenic non-CF (C38 and 16HBE14o- S1) airway epithelial cell lines to investigate whether AZM could reduce tumor necrosis factor alpha (TNF-alpha) mRNA and protein levels by real-time quantitative PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. We studied the effects on the DNA binding of NF-kappaB and specificity protein 1 (Sp1) by an ELISA. Non-CF cells express significantly lower TNF-alpha mRNA and protein levels than an isogenic CF cell line. In CF cells, AZM treatment causes a 30% reduction of TNF-alpha mRNA levels (P < 0.05) and a 45% decrease in TNF-alpha secretion (P < 0.05), reaching approximately the levels of the untreated isogenic non-CF cells. In CF cells, NF-kappaB and Sp1 DNA binding activities were also significantly decreased (about 45 and 60%, respectively; P < 0.05) after AZM treatment. Josamycin, a macrolide lacking clinically described anti-inflammatory effects, was ineffective. Finally, AZM did not alter the mRNA expression levels of interleukin-6, a proinflammatory molecule not differentially expressed in CF and isogenic non-CF cells. The results of our study support the anti-inflammatory activities of this macrolide, since we show that AZM reduced the levels of TNF-alpha and propose inhibitions of NF-kappaB and Sp1 DNA binding as possible mechanisms of this effect.     

An immunocytochemical assay to detect human CFTR expression following gene transfer

BackgroundTo assess gene therapy treatment for cystic fibrosis (CF) in clinical trials it is essential to develop robust assays that can accurately detect transgene expression in human airway epithelial cells. Our aim was to develop a reproducible immunocytochemical assay for human CFTR protein which can measure both endogenous CFTR levels and augmented CFTR expression after gene delivery.MethodsWe characterised an antibody (G449) which satisfied the criteria for use in clinical trials. We optimised our immunocytochemistry method and identified G449 dilutions at which endogenous CFTR levels were negligible in CF samples, thus enhancing detection of transgenic CFTR protein. After developing a transfection technique for brushed human nasal epithelial cells, we transfected non-CF and CF cells with a clinically relevant CpG-free plasmid encoding human CFTR.ResultsThe optimised immunocytochemistry method gave improved discrimination between CF and non-CF samples. Transfection of a CFTR expression vector into primary nasal epithelial cells resulted in detectable RNA and protein expression. CFTR protein was present in 0.05–10% of non-CF cells and 0.02–0.8% of CF cells.ConclusionWe have developed a sensitive, clinically relevant immunocytochemical assay for CFTR protein and have used it to detect transgene-expressed CFTR in transfected human primary airway epithelial cells.     

Resistance mechanisms of multiresistant Pseudomonas aeruginosa strains from Germany and correlation with hypermutation

In this study, we analyzed the mechanisms of multiresistance for 22 clinical multiresistant and clonally different Pseudomonas aeruginosa strains from Germany. Twelve and 10 strains originated from cystic fibrosis (CF) and non-CF patients, respectively. Overproduction of the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM was studied. Furthermore, loss of OprD, alterations in type II topoisomerases, AmpC overproduction, and the presence of 25 acquired resistance determinants were investigated. The presence of a hypermutation phenotype was also taken into account. Besides modifications in GyrA (91%), the most frequent mechanisms of resistance were MexXY-OprM overproduction (82%), OprD loss (82%), and AmpC overproduction (73%). Clear differences between strains from CF and non-CF patients were found: numerous genes coding for aminoglycoside-modifying enzymes and located, partially in combination with beta-lactamase genes, in class 1 integrons were found only in strains from non-CF patients. Furthermore, multiple modifications in type II topoisomerases conferring high quinolone resistance levels and overexpression of MexAB-OprM were exclusively detected in multiresistant strains from non-CF patients. Correlations of the detected phenotypes and resistance mechanisms revealed a great impact of efflux pump overproduction on multiresistance in P. aeruginosa. Confirming previous studies, we found that additional, unknown chromosomally mediated resistance mechanisms remain to be determined. In our study, 11 out of 12 strains and 3 out of 10 strains from CF patients and non-CF patients, respectively, were hypermutable. This extremely high proportion of mutator strains should be taken into consideration for the treatment of multiresistant P. aeruginosa.     

SELDI-TOF MS whole serum proteomic profiling with IMAC surface does not reliably detect prostate cancer

BACKGROUND: The analysis of bodily fluids using SELDI-TOF MS has been reported to identify signatures of spectral peaks that can be used to differentiate patients with a specific disease from normal or control patients. This report is the 2nd of 2 companion articles describing a validation study of a SELDI-TOF MS approach with IMAC surface sample processing to identify prostatic adenocarcinoma. METHODS: We sought to derive a decision algorithm for classification of prostate cancer from SELDI-TOF MS spectral data from a new retrospective sample cohort of 400 specimens. This new cohort was selected to minimize possible confounders identified in the previous study described in the companion paper. RESULTS: The resulting new classifier failed to separate patients with prostate cancer from biopsy-negative controls; nor did it separate patients with prostate cancer with Gleason scores <7 from those with Gleason scores > or =7. CONCLUSIONS: In this, the 2nd stage of our planned validation process, the SELDI-TOF MS-based protein expression profiling approach did not perform well enough to advance to the 3rd (prospective study) stage. We conclude that the results from our previous studies-in which differentiation between prostate cancer and noncancer was demonstrated-are not generalizable. Earlier study samples likely had biases in sample selection that upon removal, as in the present study, resulted in inability of the technique to discriminate cancer from noncancer cases.     

Mass spectrometry-based hepcidin measurements in serum and urine: analytical aspects and clinical implications

BACKGROUND: Discovery of the central role of hepcidin in body iron regulation has shed new light on the pathophysiology of iron disorders. Information is lacking on newer analytical approaches to measure hepcidin in serum and urine. Recent reports on the measurement of urine and serum hepcidin by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) necessitate analytical and clinical evaluation of MS-based methodologies. METHODS: We used SELDI-TOF MS, immunocapture, and tandem MS to identify and characterize hepcidin in serum and urine. In addition to diagnostic application, we investigated analytical reproducibility and biological and preanalytical variation for both serum and urine on Normal Phase 20 and Immobilized Metal Affinity Capture 30 ProteinChip arrays. We obtained samples from healthy controls and patients with documented iron-deficiency anemia, inflammation-induced anemia, thalassemia major, and hereditary hemochromatosis. RESULTS: Proteomic techniques showed that hepcidin-20, -22, and -25 isoforms are present in urine. Hepcidin-25 in serum had the same amino acid sequence as hepcidin-25 in urine, whereas hepcidin-22 was not detected in serum. The interarray CV was 15% to 27%, and interspot CV was 11% to 13%. Preliminary studies showed that hepcidin-25 differentiated disorders of iron metabolism. Urine hepcidin is more affected by multiple freeze-thaw cycles and storage conditions, but less influenced by diurnal variation, than is serum hepcidin. CONCLUSION: SELDI-TOF MS can be used to measure hepcidin in both serum and urine, but serum requires a standardized sampling protocol.     

脓毒症患者全血中钙结合蛋白S100A12的基因表达水平研究

目的检测脓毒症患者全血中钙结合蛋白S100A12的基因表达情况,并将其与炎症反应性疾病的其他炎性指标进行比较,探讨其是否可以作为脓毒症患者诊断的另一种新的指示蛋白。方法实时定量PCR方法检测脓毒症患者44例,以及10例健康人全血中S100A12基因mRNA表达水平,运用2^-△△CT算法计算相对表达量,并将S100A12的基因表达水平与白细胞、中性粒细胞绝对值、C反应蛋白、降钙素原水平进行相关性分析。结果在44例脓毒症患者中有15例患者全血中S100A12的基因表达水平升高,高表达率为34．09％;29例病人S100A12的基因表达呈低表达水平,低表达率为65．91％;S100A12的基因表达水平与白细胞、中性粒细胞绝对值、c反应蛋白、降钙素原水平经相关性分析结果显示P值均大于0．05,差异无统计学意义。结论尚不能认为在脓毒症患者全血中S100A12普遍呈高表达状态,暂不能将其作为脓毒症的另一种新的指示蛋白。