Jos Hermans

Prediction of relapses in Wegener's granulomatosis by measurement of antineutrophil cytoplasmic antibody levels: a prospective study.

OBJECTIVE Prediction of relapses in Wegeners granulomatosis (WG) by measuring levels of antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) or myeloperoxidase (MPO) remains a controversial issue. To assess the value of serial quantification of ANCA by indirect immunofluorescence (IIF) and antigen-specific enzyme-linked immunosorbent assay (ELISA) for monitoring disease activity in patients with WG, a prospective observational study was conducted in patients with WG attending an outpatient clinic in the Netherlands. METHODS One hundred patients with WG (85 with PR3-ANCA, 15 with MPO-ANCA) were studied prospectively from 1996 to 1998. Serum samples were obtained and analyzed every 2 months for ANCA levels. Disease activity was prospectively assessed without knowledge of the ANCA levels. RESULTS Relapses occurred in 37 of 100 patients (37%). Thirty-four (92%) of the 37 patients showed a rise in the level of ANCA preceding their relapse, as detected by ELISA or IIF. The predictive value of an increase in ANCA titers for relapse was 57% (17 of 30) for cytoplasmic/classic ANCA (cANCA; by IIF), 71% (27 of 38) for PR3-ANCA (by ELISA), and 100% (3 of 3) for MPO-ANCA (by ELISA). The predictive value of a rise in ANCA as measured by ELISA or IIF did not substantially improve following concomitant measurement of the IgG3 subclass of PR3-ANCA. Forty-three percent of patients who showed a rise in cANCA (by IIF) and 29% with a rise in PR3-ANCA (by ELISA) did not subsequently experience a relapse. CONCLUSION Serial measurement of ANCA levels is valuable for the early prediction of relapses in patients with WG.

Leukocyte activation in sepsis; correlations with disease state and mortality

Objective: The immune response in sepsis shows a bimodal pattern consisting of an early, frequently exaggerated inflammatory response followed by a state of hyporesponsiveness often referred to as the compensatory anti-inflammatory response syndrome (CARS). Insight into the disease state may be helpful in deciding whether to choose immune stimulatory or anti-inflammatory therapy in these patients and may determine clinical outcome. We hypothesized that poor outcome in patients with sepsis is related to the severity of CARS, as reflected in the degree of leukocyte activation. Design: Prospective study. Setting: Intensive and respiratory care unit at a university hospital. Patients: Twenty consecutive patients with sepsis and 20 healthy age-matched volunteers. Interventions: None. Measurements and results: Analysis of surface expression of HLA-DR, CD11 b, ICAM-1, CD66 b, CD63 and CD64 on neutrophils and monocytes by flow cytometry and determination of plasma concentrations of lactoferrin, interleukin 6 and neopterin by ELISA at the time of diagnosis. Patient data were related to those of controls; moreover patient data between survivors and non-survivors were compared. Increased expression of all markers, except HLA-DR, was observed on both neutrophils and monocytes from patients compared to healthy controls. HLA-DR expression on monocytes was significantly decreased in patients with sepsis (p < 0.01). Expression of CD11 b and HLE on neutrophils, and ICAM-1 on monocytes, were lower in patients who died compared to those who survived (p < 0.05). Conclusion: In sepsis, both neutrophils and monocytes are activated compared to healthy controls. Poor prognosis is associated with a lower expression of activation markers on monocytes and neutrophils, suggesting that poor outcome in these patients may be due to the compensatory anti-inflammatory response.

Gastric low-grade MALT lymphoma, high-grade MALT lymphoma and diffuse large B cell lymphoma show different frequencies of trisomy.

Gastric MALT lymphoma is a distinct entity related to Helicobacter pylori gastritis. Some studies suggest a role for trisomy 3 in the genesis of these lymphomas, but they mainly focused on low-grade MALT lymphoma. Gastric MALT lymphoma, however, comprises a spectrum from low- to high-grade cases. Furthermore, its exact relation to primary diffuse large B cell lymphoma (DLBCL) of the stomach is not clear. We applied in situ hybridisation (ISH) with centromeric probes on 43 samples of 39 patients with primary gastric lymphoma (13 samples with low-grade MALT lymphoma, 25 with high-grade MALT lymphoma and five with DLBCL) to detect numerical aberrations of 10 chromosomes. ISH was performed immunohistochemically on nuclei isolated from paraffin-embedded resection tissue and on whole paraffin sections using immunofluorescence. In six of 13 low-grade MALT lymphomas trisomy was detected (46%) and mostly involved chromosome 3 (33%). In high-grade MALT lymphomas, trisomies were found in 16 of 25 cases (64%), mainly involving chromosomes 12 and 18. Trisomy 3 was present in only 13% of these cases. Of five DLBCL, only one showed trisomy. Nine of the 16 aberrant high-grade MALT lymphomas (56%) showed trisomy of more than one chromosome per case vs two of six for low-grade cases. In lymphomas with separate low- and high-grade tumour components some trisomies were detected in both components, whereas others occurred only in the high-grade tumour cells. This supports the hypothesis that high-grade MALT lymphomas can develop from a low-grade type and that this progression is accompanied by the acquisition of more genetic aberrations. However, trisomy 3 probably does not play a major role in this progression.

Impact of KRAS and TP53 mutations on survival in patients with left- and right-sided Dukes' C colon cancer

OBJECTIVE:It has been suggested that KRAS and TP53 mutated tumors might influence the phenotypic behavior of left- and right-sided colon tumors. We investigated the incidence of these mutations in left- and right-sided colon tumors and their possible influence on survival in a homogeneous group of patients with Dukes’ C colon cancers.METHODS:The primary tumors of 55 patients with a sporadic Dukes’ C colon cancer, all treated with adjuvant chemotherapy were analyzed for the presence of KRAS and TP53 mutations. Mutation detection of the KRAS and TP53 genes was performed on paraffin-embedded tumor material, using denaturating gradient gel electrophoresis. The 5-yr survival rates of KRAS and TP53 mutated tumors were analyzed regarding right-sided tumors (defined as tumors up to the splenic flexure) and left-sided tumors (defined as tumors from the splenic flexure to the rectosigmoid peritoneal reflection).RESULTS:KRAS mutations occurred more frequently in the right colon compared to the left colon (R = 38% (10/26); L = 10% (3/29); χ2 test: p = 0.014). KRAS mutations did not influence survival in patients with right-sided colon tumors. Patients with KRAS mutation–negative tumors in the right colon, however, had a significantly worse survival than patients with left-sided KRAS mutation–negative tumors (5-yr survival; R: 34% vs L: 65%, log-rank test: p = 0.007). TP53 mutations of a possible causative nature were found in 24 tumors (44%). Neither the incidence (R = 42% (11/26); L = 45% (13/29)) nor the survival of TP53 mutated tumors differed significantly between left- and right-sided tumors. Furthermore, survival of patients with TP53 mutation–negative tumors did not differ significantly between left- and right-sided tumors.CONCLUSIONS:There seems to be no difference in survival rate between patients with KRAS mutated and KRAS negative Dukes’ C colon tumors; however, KRAS mutations are more frequently found in the right colon compared to the left colon. TP53 mutations do not have predominance for either side of the colon, and there are no differences in survival in patients with left-sided versus right-sided tumors. Patients with KRAS-nonmutated tumors in the right colon did have a worse survival compared to those with such tumors in the left colon. This suggests that other genetic factors may play a role in tumor genesis in this subgroup of patients.

Prognostic significance of K-ras and TP53 mutations in the role of adjuvant chemotherapy on survival in patients with Dukes C colon cancer

PURPOSE: Mutations in K-ras andTP53 genes are common in colorectal cancer. They affect biologic behavior and might influence chemotherapy susceptibility in these tumors. We investigated whether the survival of patients with Dukes C colon cancer treated with adjuvant chemotherapy is influenced by K-ras andTP53 mutations. METHODS: Mutation screening of the hot spots of the K-ras gene and of the evolutionarily conserved regions of theTP53 gene was performed by denaturing gradient gel electrophoresis technique in formalin-fixed paraffin-embedded specimens of 55 consecutive patients with Dukes C colon cancer treated with adjuvant 5-fluorouracil-based chemotherapy. The median follow-up was 47 (range, 32–66) months. RESULTS: Alterations in the mutation hot spots of K-ras were found at codon 12 (n=11) and 13 (n=4) in 15 of the 55 carcinomas (27 percent). No mutation was found at codon 61. Mutations of a probably causative nature in the evolutionarily conserved regions (exons 5–8) of theTP53 gene were found in 24 tumors (44 percent). K-ras andTP53 mutations were found equally in the group with recurrent disease (7/26 (26 percent) and 12/27 (44 percent), respectively) and in the group without recurrences (8/28 (24 percent) and 12/28 (43 percent), respectively). Cancer-specific survival did not differ significantly between patients with K-ras orTP53 or both mutated and nonmutated tumors, respectively (log-rank test: K-ras, P=0.72 andTP53, P=0.77; K-ras andTP53, P=0.8). Also, potentially aggressive K-ras codon 12 and 13 mutations had the same survival as tumors without these mutations (log-rank test;P=0.73). CONCLUSIONS: Patients with K-ras orTP53 or both mutated Dukes C colon tumors have the same survival as nonmutated tumors when treated with adjuvant chemotherapy. These data suggest that mutations in K-ras orTP53 alone are not prognostic indicators in patients with Dukes C colon cancer receiving adjuvant 5-Fluorouracil-based therapy.

Quantification of free and total desmosine and isodesmosine in human urine by liquid chromatography tandem mass spectrometry: A comparison of the surrogate-analyte and the surrogate-matrix approach for quantitation

In spite of the data suggesting the potential of urinary desmosine (DES) and isodesmosine (IDS) as biomarkers for elevated lung elastic fiber turnover, further validation in large-scale studies of COPD populations, as well as the analysis of longitudinal samples is required. Validated analytical methods that allow the accurate and precise quantification of DES and IDS in human urine are mandatory in order to properly evaluate the outcome of such clinical studies. In this work, we present the development and full validation of two methods that allow DES and IDS measurement in human urine, one for the free and one for the total (free+peptide-bound) forms. To this end we compared the two principle approaches that are used for the absolute quantification of endogenous compounds in biological samples, analysis against calibrators containing authentic analyte in surrogate matrix or containing surrogate analyte in authentic matrix. The validated methods were employed for the analysis of a small set of samples including healthy never-smokers, healthy current-smokers and COPD patients. This is the first time that the analysis of urinary free DES, free IDS, total DES, and total IDS has been fully validated and that the surrogate analyte approach has been evaluated for their quantification in biological samples. Results indicate that the presented methods have the necessary quality and level of validation to assess the potential of urinary DES and IDS levels as biomarkers for the progression of COPD and the effect of therapeutic interventions.

Suggestions for the optimization of the initial tobramycin dose in adolescent and adult patients with cystic fibrosis

Clinical pharmacokinetic data of intravenously administered tobramycin in 34 patients with cystic fibrosis (CF) were correlated with patient parameters. Patients began tobramycin therapy with 10 mg/kg/day in three divided doses. Peak and trough serum concentrations were measured. Tobramycin dose was adjusted to a 30 min postdose peak of 10 mg/L and a predose trough of 1 mg/L. Pharmacokinetic data were calculated according to a one-compartment open model and were correlated with clinical data. Tobramycin half-life and total body clearance did not correlate with age, actual body weight, lean body mass, height, or body surface area. Tobramycin volume of distribution correlated with actual body weight (p < 0.02), lean body mass (p < 0.002), height (p < 0.05), and body surface area (p < 0.01), but not with age. The required daily dose after adjustment to a peak serum concentration of 10 mg/L and a trough level of 1 mg/L correlated with lean body mass (p < 0.02) and body surface area (p < 0.05). Based on our findings, the initial daily dose of tobramycin in patients with CF should be calculated by lean body mass rather than actual body weight or body surface area. A formula is presented to calculate the initial daily dose of tobramycin in CF patients who have normal renal function. Monitoring of tobramycin serum levels remains, however, necessary.

Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum

The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous quantification of the 60-kDa biopharmaceutical proteins recombinant human tumor necrosis factor-related apoptosis-inducing ligand wild type (rhTRAIL(WT)) and its death receptor 4 (DR4)-specific variant rhTRAIL(4C7) in human and mouse serum. Selective enrichment of TRAIL was accomplished by immobilized metal affinity chromatography (IMAC), which was followed by tryptic digestion of the enriched sample and quantification of a suitable signature peptide. For absolute quantification, (15)N-metabolically labeled internal standards of rhTRAIL(WT) and rhTRAIL(4C7) were used. Since the signature peptides that provided the highest sensitivity and allowed discrimination between rhTRAIL(WT) and rhTRAIL(4C7) contained methionine residues, we oxidized these quantitatively to their sulfoxides by the addition of 0.25% (w/w) hydrogen peroxide. The final method has a lower limit of quantification of 20 ng/mL (ca. 350 pM) and was fully validated according to current international guidelines for bioanalysis. To show the applicability of the LC-MS/MS method for pharmacokinetic studies, we quantified rhTRAIL(WT) and rhTRAIL(4C7) simultaneously in serum from mice injected intraperitoneally at a dose of 5 mg/kg for each protein. This is the first time that two variants of rhTRAIL differing by only a few amino acids have been analyzed simultaneously in serum, an approach that is not possible by conventional enzyme-linked immuno-sorbent assay (ELISA) analysis.

Quantification of matrix metalloprotease-9 in bronchoalveolar lavage fluid by selected reaction monitoring with microfluidics nano-liquid-chromatography-mass spectrometry

Quantitative protein analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the selected reaction monitoring (SRM) mode was used to quantify matrix metalloprotease-9 (MMP-9; ∼90 kDa) in bronchoalveolar lavage fluid (BALF) from patients having undergone lung transplantation. We developed an SRM assay for microfluidics-based nanoLC-MS/MS on a triple quadrupole mass spectrometer based on two signature peptides. Samples were prepared by chloroform-methanol precipitation followed by trypsin digestion in the presence of stable-isotope-labeled internal peptide standards. The method allows accurate quantification of MMP-9 in BALF with an LLOQ of 2.9 ng/mL and an LLOD of 0.25 ng/mL without the use of extensive fractionation or antibodies.

Electron Transfer and Collision Induced Dissociation of Non-Derivatized and Derivatized Desmosine and Isodesmosine

AbstractElectron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH3, CO, H2O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples. FigureA mechanistic study of electron transfer dissociation (ETD) and collision-induced dissociation (CID) of labeled and free desmosine and isodesmosine provides evidence that CID is mainly due to charge-directed fragmentation while ETD is initiated by the generation of a hydrogen atom at the initial protonation site, and its subsequent transfer to the pyridinium ring.