Targeted identification of infection-related HLA class I-presented epitopes by stable isotope tagging of epitopes (SITE).

Identification of peptides presented in human leukocyte antigen (HLA) class I molecules after viral infection is of strategic importance for immunology and vaccine development. A powerful strategy aimed at the rapid, unambiguous identification of naturally processed HLA class I-associated peptides, which are induced by viral infection, is presented here. The methodology, stable isotope tagging of epitopes (SITE), is based on metabolic labeling of endogenously synthesized proteins during infection. This is accomplished by culturing virus-infected cells with stable isotope-labeled amino acids that are expected to be anchor residues for the human leukocyte antigen allele of interest. Subsequently, these cells are mixed with an equal number of noninfected cells, which are cultured in normal medium. Finally, peptides are acid-eluted from immunoprecipitated HLA molecules and subjected to two-dimensional nanoscale liquid chromatography-mass spectrometry analysis. Virus-induced peptides are identified through computer-assisted detection of characteristic, binomially distributed ratios of labeled and unlabeled molecules.

Topical negative pressure therapy in wounds after cardiothoracic surgery: successful experience supported by literature.

BACKGROUND: Patients undergoing cardiothoracic surgery are at substantial risk of developing surgical site infections (SSI). SSI is not only associated with an increased morbidity but also with high mortality. Topical negative pressure therapy (TNP) is a promising method for treating surgical site defects (SSD). In recent years, we have gained a wide experience with TNP in a great variety of SSD. METHODS: We completed a prospective follow-up report of all patients treated with TNP after cardiothoracic surgery at the Academic Medical Centre Amsterdam, a university hospital. A review of the current evidence for TNP in cardiothoracic surgery is presented. RESULTS: Between August 2000 and March 2005, TNP was used in 105 patients in 113 SSD. As we gained more experience, we saw a decline in hospital stay ( P < 0.0001) and duration of TNP therapy. Surgical closure was performed in 62 % of patients using simple surgical (reconstructive) techniques. Therapy-related complications were rare (n = 1). CONCLUSION: Based on clinical findings and supported by the research presented, the treatment modality of choice for SSD after cardiothoracic surgery is TNP.

Risk control of surgical site infection after cardiothoracic surgery.

The purpose of this prospective study was to investigate whether a risk control programme based on risk assessment, new treatment modalities and the presence of a surveillance programme reduces the incidence of surgical site infections (SSI). Between January 2001 and December 2003, 167 patients were treated for a total of 183 SSIs. Data were collected on pre-operative risk factors, intra-operative data and postoperative recovery, including complications, infecting organisms, SSI treatment techniques and length of hospital stay. In this series, the total incidence of SSI was 5.6%. The mean age of affected patients was 65.1 years with a range of 20-87 years. Mean intensive care and hospital stay for SSI was 3.6 days and 18.8 days, respectively. Total mortality was 4.8%. Many risk factors were encountered, some of which were associated with a high morbidity. The majority of SSIs were treated by topical negative pressure therapy (N=81), which gave few side-effects and good clinical results. After starting the surveillance programme, a steady decline in prevalence was observed from 8.9% to 3.9%. This series adds to the evidence that SSI after cardiothoracic surgery is a major but mainly preventable cause of morbidity and mortality. Risk factor assessment, application of novel treatment modalities and an adequate surveillance system all increased patient safety.

High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation.

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.

Differential protein expression in phenotypic variants of Streptococcus pneumoniae.

Streptococcus pneumoniae undergoes spontaneous phase variation resulting in opaque and transparent colony forms. Differences in colony opacity correlate with differences in virulence: the transparent variants are more capable of colonizing the nasopharynx, whereas the opaque variants show increased virulence during systemic infections. To gain insight into the pathogenesis of pneumococcal disease at the molecular level, protein expression patterns of the phenotypic variants of two pneumococcal strains were compared by high-resolution two-dimensional protein electrophoresis. In comparison with transparent variants, the opaque variants reduced the expression of two proteins and overexpressed one protein. The proteins were identified by mass spectrometric analysis. The protein overexpressed in the opaque phenotype revealed significant homology to elongation factor Ts of Helicobacter pylori. One of the two proteins that were underexpressed in the opaque variants revealed significant homology to the proteinase maturation protein PrtM of Lactocobacillus paracasei, a member of the family of peptidyl-prolyl cis/trans isomerases. A consensus lipoprotein signal sequence suggests that the putative proteinase maturation protein A, designated PpmA, is located at the surface of the pneumococcus and may play a role in the maturation of surface or secreted proteins. The second underexpressed protein was identified as pyruvate oxidase, SpxB. The lower SpxB expression in opaque variants most probably explains the reduced production of hydrogen peroxide, a reaction product of SpxB, in this variant. Since a spxB-defective pneumococcal mutant has decreased ability to colonize the nasopharynx (B. Spellerberg, D. R. Cundell, J. Sandros, B. J. Pearce, I. Idanpaan-Heikkila, C. Rosenow, and H. R. Masure, 1996. Mol. Microbiol. 19:803-813, 1996), our data suggest that SpxB plays an important role in enhancing the ability of transparent variants to efficiently colonize the nasopharynx.

The putative proteinase maturation protein A of Streptococcus pneumoniae is a conserved surface protein with potential to elicit protective immune responses.

Surface-exposed proteins often play an important role in the interaction between pathogenic bacteria and their host. We isolated a pool of hydrophobic, surface-associated proteins of Streptococcus pneumoniae. The opsonophagocytic activity of hyperimmune serum raised against this protein fraction was high and species specific. Moreover, the opsonophagocytic activity was independent of the capsular type and chromosomal genotype of the pneumococcus. Since the opsonophagocytic activity is presumed to correlate with in vivo protection, these data indicate that the protein fraction has the potential to elicit species-specific immune protection with cross-protection against various pneumococcal strains. Individual proteins in the extract were purified by two-dimensional gel electrophoresis. Antibodies raised against three distinct proteins contributed to the opsonophagocytic activity of the serum. The proteins were identified by mass spectrometry and N-terminal amino acid sequencing. Two proteins were the previously characterized pneumococcal surface protein A and oligopeptide-binding lipoprotein AmiA. The third protein was the recently identified putative proteinase maturation protein A (PpmA), which showed homology to members of the family of peptidyl-prolyl cis/trans isomerases. Immunoelectron microscopy demonstrated that PpmA was associated with the pneumococcal surface. In addition, PpmA was shown to elicit species-specific opsonophagocytic antibodies that were cross-reactive with various pneumococcal strains. This antibody cross-reactivity was in line with the limited sequence variation of ppmA. The importance of PpmA in pneumococcal pathogenesis was demonstrated in a mouse pneumonia model. Pneumococcal ppmA-deficient mutants showed reduced virulence. The properties of PpmA reported here indicate its potential for inclusion in multicomponent protein vaccines.

A single naturally processed measles virus peptide fully dominates the HLA-A*0201-associated peptide display and is mutated at its anchor position in persistent viral strains.

We studied the natural MHC class I display of measles virus (MV) epitopes. Peptide ligands associated with HLA-A*0201 were purified from a B lymphoblastoid cell line prior to and after infection with MV. Infection-induced peptides were revealed using microcapillary reversed phase high performance liquid chromatography electrospray ionization/mass spectrometry (microLC-ESI/MS) by subtraction of the "infected" and "uninfected" ion traces. Three naturally processed viral epitopes derived from different MV proteins were identified through tandem MS sequencing. These peptides were expressed at widely divergent levels of HLA-peptide complexes, but had similar binding capacities to HLA-A*0201. The most abundant viral peptide species, identified as residues 84-92 (KLWESPQEI) of the MV nonstructural C protein, was expressed at an unprecedented high density (> 10(5) copies per cell) and was immunogenic in HLA-A2/Kb-transgenic mice. Furthermore, natural mutants of this epitope, occurring in persistent lethal MV strains, were shown to have lost their HLA-A*0201 binding capacity. Thus, here we report for the first time the direct discovery through microLC-ESI/MS of a uniquely dominant viral HLA class I ligand, KLWESPQEI, with features eligible for immune selection pressure.

Sheathless preconcentration-capillary zone electrophoresis-mass spectrometry applied to peptide analysis.

A reliable capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry system has been developed for the sensitive analysis and sequencing of peptides. The system comprises (1) a zero dead volume on-line sample preconcentration interface allowing over 100-fold increase in sample volume introduction and (2) a zero-dead volume, durable electrospray emitter yielding stable, low background electrospray signals, both proving not to compromize the electrophoretic performance. Sub-fmol sensitivities were obtained in applications to complex peptide samples derived from tryptic digestion of proteins and naturally processed major histocompatibility complex class I associated peptides.

Enzyme amplification as detection tool in continuous-flow systems. I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis.

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.

Enzyme amplification as detection tool in continuous-flow systems. II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin.

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compounds were biotinylated off-line with biotin aminocaproic hydrazide (BACH). On-column biotinylation was performed by preconcentration of anthracene aldehyde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affinity to biotin, was added post-column to the HPLC effluent. Excess of enzyme-labeled affinity protein was removed by means of an immobilized biotin column. After separation of free and bound fraction, substrate was added, which was converted to a fluorescent product by the enzyme label. Using alkaline phosphatase as an enzyme label, a mass detection limit after on-column preconcentration and biotinylation of 250 fmol was achieved.

Isolation, identification and immunosuppressive activity of a new IMM-125 metabolite from human liver microsomes. Identification of its cyclophilin A-IMM-125 metabolite complex by nanospray tandem mass spectrometry.

The isolation from human liver microsomes and identification by electrospray mass spectrometry and tandem mass spectrometry of a new metabolite of IMM-125 resulting from the biotransformation of the amino acid 1 vinylic methyl group to a carboxylic acid, called the IMM-125-COOH metabolite, is described. It was found that the complex of this new metabolite with cyclophilin A is formed less easily than the corresponding cyclophilin A-IMM-125-CH2OH main metabolite and cyclophilin A-IMM-125 complexes. However, when formed, the IMM-125-COOH metabolite-cyclophilin A complex requires more collision-induced dissociation (CID) to dissociate the complex than the complexes formed with the two other ligands. The nanospray tandem mass spectrum of the IMM-125-COOH metabolite-cyclophilin A complex (m/z 1755) gives rise to cyclophilin A-ligand complexes of m/z 1751 by elimination of CO2 and of m/z 1749 by loss of CO2 and H2O or glycerol. Since immunosuppressive activity is known to be dependent on the formation of a binary complex between cyclophilin A and the drug and since the target for the binary complex was found to be the calcium- and calmodulin-dependent protein phosphatase, calcineurin, it could be interesting to measure for structurally related immunosuppressive drugs the CID energy necessary to dissociate the binary complexes in order to evaluate whether a correlation with the phosphatase activity could be derived.

A microcapillary column switching HPLC-electrospray ionization MS system for the direct identification of peptides presented by major histocompatibility complex class I molecules.

A microcapillary column switching high-performance liquid chromatography (HPLC) system was developed for the separation of major histocompatibility complex (MHC) class I associated peptides. Combination of the column switching system with electrospray ionization mass spectrometry (ESIMS) enabled the detection and identification of the peptides at low-femtomole levels. Sample volumes of 30-50 microL were injected and concentrated onto a short, 100-micron-i.d. precolumn. The precolumn was coupled to a 100-micron-i.d. reversed-phase analytical HPLC column via a six-port valve. Peptides were separated on the analytical column using an ESI-compatible mobile phase at a flow rate of 0.5 microL/min. Peptides were eluted directly into the ESI source of either a magnetic sector MS or an ion trap MS. Peptides associated with human leukocyte antigen A*0201 molecules were determined in immunoaffinity-purified extracts from either measles virus infected cells or uninfected cells by microcapillary column switching HPLC-ESIMS. The approach toward detection of virus-specific peptides we used was based on the comparison of ion chromatograms obtained from the LC-MS analysis of extracts from virally infected cells and their uninfected counterparts. In this way, the molecular mass of peptides unique to virus infected cells was obtained. The utility of the system is demonstrated by the identification of a candidate epitope. Microcapillary column switching HPLC was used along with ESI ion trap tandem MS to identify the naturally processed viral peptide KLWESPQEI. This peptide was found to derive from the measles virus nonstructural protein C. The approach described here provides a versatile and sensitive method for the direct identification of viral peptides associated with MHC class I molecules.

A new design for large, dilute sample loading in capillary electrophoresis.

In this paper, we report on a new design for a fully in-line sample preconcentration for capillary zone electrophoresis (CZE). It comprises a miniaturized (50-75 microns i.d. x 1 mm) reversed-phase C18 bed (2-4 nL) connected without any dead volume to a CZE capillary. The system permits the fast loading of relatively large sample volumes into the CZE column after elution of the trapped analytes with a small organic solvent volume (20-50 nL). The trapping capacity for peptides was in the high picomole range. No breakthrough or other losses were observed either during microliter-range injections or subsequent washings. The trapped analytes were eluted into the CZE column with less than 50 nL methanol/water (80:2 vol/vol). The narrow band in which samples enter the CZE columns allows direct CZE separation without the necessity of refocusing or stacking. The system performance was studied in relation to the volume and concentration of the leading stacking buffer using electrospray ionization (ESI)-compatible electrolytes. In addition, the system allows fractionation of the trapped analytes using subsequent stronger elutropic elution organic solvent/water mixtures, resulting in a semi-2D separation technique. The system was applied to the analysis of protein digest mixtures and to extremely complex major histocompatibility complex (MHC) class I associated peptides.

Clinical evaluation of Duraflo II heparin treated extracorporeal circulation circuits (2nd version). The European Working Group on heparin coated extracorporeal circulation circuits.

OBJECTIVES: To evaluate whether the application of heparin treated circuits for elective coronary artery surgery improves postoperative recovery, a European multicenter randomised clinical trial was carried out. METHODS: In 11 European heart centers, 805 low-risk patients underwent cardiopulmonary bypass (CPB) with either an untreated circuit (n = 407) or an identical but heparin treated circuit (n = 398, Duraflo II). RESULTS: Significant differences were found among participating centers with respect to patient characteristics, blood handling procedures and postoperative care. The use of heparin treated circuits revealed no overall changes in blood loss, blood use, time on ventilator, occurrence of adverse events, morbidity, mortality, and intensive care stay. These results did not change after adjustment for centers and (other) prognostic factors as analysed with logistic regression. In both groups no clinical or technical (patient or device related) side effects were reported. Because female gender and aortic cross clamp time appeared as prognostic factors in the logistic regression analysis, a subgroup analysis with these variables was performed. In a subpopulation of females (n = 99), those receiving heparin treated circuits needed less blood products, had a lower incidence of rhythm disturbances and were extubated earlier than controls. In another subgroup of patients with aortic cross clamp time exceeding 60 min (n = 197), the amount of patients requiring prolonged intensive care treatment (> 24 h) was significantly lower when they received heparin treated circuits versus controls. CONCLUSION: These findings suggest that improved recovery can be expected with heparin treated circuits in specific higher risk patient populations (e.g. females) and when prolonged aortic cross clamp time is anticipated. Further investigations are recommended to analyses the clinical benefit of heparin treated circuits in studies with patients in different well defined risk categories and under better standardised circumstances.

On-line derivatization of peptides for improved sequence analysis by micro-column liquid chromatography coupled with electrospray ionization-tandem mass spectrometry.

An on-line method has been developed for the derivatization and coupled liquid chromatography (LC)/electrospray ionization (ESI) MS analysis of peptides at the femtomol level. Peptides are reacted with N-succinimidyl-2(3-pyridyl)acetate (SPA) in buffered aqueous medium at pH7 following loading on a precolumn (PC) in a microcolumn switching system. The fast-hydrolysing reagent is dissolved in dry methanol and mixed, in a 3 vol% ratio, with a buffer just before reaching the sample on the PC. Following the reaction and wash, the N-pyridylacetyl (PA) derivatives formed are transferred to the analytical micro-high-performance (HP) LC column for separation and subsequent ESI tandem MS analysis. The reaction is nearly quantitative and takes place selectively at the N-terminal and lysine amino functions, the latter providing a chemical means to distinguish between isobaric residues lysine and glutamine. In some cases, a minor reaction was observed with the tyrosine hydroxyl group. The N-terminal PA group was able to alter the collision-induced fragmentation pathway of peptides in favour of the formation of abundant b-type ions, a helpful feature for sequence elucidation of unknown peptides, particularly with quadrupole ion trap instruments.

Screening and analysis of polar pesticides in environmental monitoring programmes by coupled-column liquid chromatography and gas chromatography-mass spectrometry.

Screening and analysis of polar pesticides based on coupled-column reversed-phase liquid chromatography (LC-LC) and GC- or LC-MS is a powerful tool in the execution of environmental monitoring programmes. This paper presents a unified approach utilising LC-LC screening followed by GC-MS confirmation. As polar pesticides are not generally amenable to GC a widely applicable derivation technique is used. The results demonstrate that the proposed LC and MS techniques are capable of analysing a wide range of polar pesticides down to levels of 0.1 microgram/l (EU limit for drinking water). LC switching techniques for group analysis or individual compounds rely on the reversed-phase retention and the UV detectability of the pesticides in combination with the choice of the LC columns. Fast miniaturised derivatization prior to GC-MS forms an integral part in the proposed strategy. In order to avoid extraction losses, derivation in the aqueous sample, preferably with electrophoric reagents with enhanced sensitivity in GC-NICI-MS are employed where possible. In this communication, method development and validation fitting in the strategy are evaluated and the results of the combined approach are discussed.

Modified sampling and analysis method for large volatility range airborne polycyclic aromatic hydrocarbons (PAH) using gas chromatography-mass spectrometry.

A sampling method has been developed for the measurement for polycyclic aromatic hydrocarbons in ambient air by gas chromatography isotope dilution mass spectrometry. The method has been designed to measure the largest possible volatility range of PAHs including the abundant naphthalenes. Sample volumes were approx. 500 m(3) in size at a sampling rate of approx. 18 m(3)/h. The sampler contained three sorption stages for the simultaneous capturing of particle bound and low and high volatile gaseous PAH, respectivley. Recoveries of sampling spikes were on average 90%. The detection limit was approx. 5 pg/m(3) for the high boiling range PAH. Results obtained showed a quite steady profile for most PAH in background air in The Netherlands. Comparison of abundance ratios with literature data indicate that traffic exhausts are the major source for the PAH in the area.

Congener-specific bioavailability of PCDD/Fs and coplanar PCBs in cows: laboratory and field measurements.

To estimate congener-specific bioavailabilities for 17 PCDD/Fs in cows grazing near a MSW incinerator both a controlled lab study and a field study were performed. In the lab study the estimates were derived from the elevated concentrations in milk from two cows after administration of a single dose of contaminated fly ash. In the field study, located near a large MSW incinerator, daily samples of grass and milk collected over a period of 60 days were pooled to two monthly bulk samples. The concentrations in these bulk samples of grass and milk were used to estimate the bioavailabilities of the 17 PCDD/Fs as well as of three coplanar PCBs. With the concentrations of PCDD/Fs expressed in I-TEQs the bioavailability in cows was estimated at +/- 7.5% in the field study. The congener-specific bioavailabilities correlated well between the two studies, as well as with previously reported values in the literature. However, the absolute levels differed considerably between studies, indicating a strong matrix effect.