A new approach to study exhaled proteins as potential biomarkers for asthma.

BACKGROUND: Asthma is a complex clinical disease characterized by airway inflammation. Recently, various studies reported on the analysis of exhaled breath condensate (EBC) in the search for potential biomarkers for asthma. However, in a complex disease such as asthma, one biomarker might not be enough for early diagnosis or follow-up. OBJECTIVE: The use of proteome analysis may reveal disease-specific proteolytic peptide or protein patterns, and may lead to the identification of novel proteins for the detection of asthma. METHODS: Liquid chromatography and mass spectrometry were used to separate and detect proteins (proteolytic peptides) present in EBC samples from 30 healthy children and 40 children with asthma in the age group of 6-12 years. RESULTS: Support vector machine analysis resulted in differentiating profiles based on asthma status. These proteolytic peptide patterns were not correlated to some well known (spirometry, exhaled nitric oxide) and more recently described exhaled markers (EBC pH, LTB4). The more abundant proteins in EBC were identified as cytokeratins, albumin, actin, haemoglobin, lysozyme, dermcidin, and calgranulin B. CONCLUSION: Although the exact role in the disease development or physiological state of the airways of the proteins described in the presented pattern is not clear at this moment, this is an important step in the search for exhaled biomarkers for asthma. This study shows that EBC contains proteins that are of interest for future non-invasive asthma diagnosis or follow-up.

Denitrification at pH 4 by a soil-derived Rhodanobacter-dominated community.

Soil denitrification is a major source of nitrous oxide emission that causes ozone depletion and global warming. Low soil pH influences the relative amount of N2O produced and consumed by denitrification. Furthermore, denitrification is strongly inhibited in pure cultures of denitrifying microorganisms below pH 5. Soils, however, have been shown to denitrify at pH values as low as pH 3. Here we used a continuous bioreactor to investigate the possibility of significant denitrification at low pH under controlled conditions with soil microorganisms and naturally available electron donors. Significant NO3⁻ and N2O reduction were observed for 3 months without the addition of any external electron donor. Batch incubations with the enriched biomass showed that low pH as well as low electron donor availability promoted the relative abundance of N2O as denitrification end-product. Molecular analysis of the enriched biomass revealed that a Rhodanobacter-like bacterium dominated the community in 16S rRNA gene libraries as well as in FISH microscopy during the highest denitrification activity in the reactor. We conclude that denitrification at pH 4 with natural electron donors is possible and that a Rhodanobacter species may be one of the microorganisms involved in acidic denitrification in soils.

Application of human CFU-Mk assay to predict potential thrombocytotoxicity of drugs.

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors, identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count, based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions, and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity, according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10, it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50, thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors.

N2O emission hotspots at different spatial scales and governing factors for small scale hotspots.

Chronically nitrate-loaded riparian buffer zones show high N(2)O emissions. Often, a large part of the N(2)O is emitted from small surface areas, resulting in high spatial variability in these buffer zones. These small surface areas with high N(2)O emissions (hotspots) need to be investigated to generate knowledge on the factors governing N(2)O emissions. In this study the N(2)O emission variability was investigated at different spatial scales. Therefore N(2)O emissions from three 32 m(2) grids were determined in summer and winter. Spatial variation and total emission were determined on three different scales (0.3 m(2), 0.018 m(2) and 0.0013 m(2)) at plots with different levels of N(2)O emissions. Spatial variation was high at all scales determined and highest at the smallest scale. To test possible factors inducing small scale hotspots, soil samples were collected for slurry incubation to determine responses to increased electron donor/acceptor availability. Acetate addition did increase N(2)O production, but nitrate addition failed to increase total denitrification or net N(2)O production. N(2)O production was similar in all soil slurries, independent of their origin from high or low emission soils, indicating that environmental conditions (including physical factors like gas diffusion) rather than microbial community composition governed N(2)O emission rates.

Cell types involved in allergic asthma and their use in in vitro models to assess respiratory sensitization.

This review first describes the mechanism and cell types involved in allergic asthma, which is a complex clinical disease characterized by airway obstruction, airway inflammation and airway hyperresponsiveness to a variety of stimuli. The development of allergic asthma exists of three phases, namely the induction phase, the early-phase asthmatic reaction (EAR) and the late-phase asthmatic reaction (LAR). In the induction phase, antigen-presenting cells play a major role. Most important cells in the EAR are mast cells, and during the LAR, various cell types, such as eosinophils, neutrophils, T cells, macrophages, dendritic cells (DCs), and cells that endow structure are involved. In occupational asthma, this immunological mechanism is involved in 90% of the cases. The second part of this review gives an overview of in vitro models to assess the hazardous potential of high- and low-molecular weight chemicals on the respiratory system. In order to develop a good in vitro model for respiratory allergy, the choice of appropriate cell types is important. Epithelial cells, macrophages and DCs are currently the most used models in this field of research.

Induction of oral tolerance to oxidized low-density lipoprotein ameliorates atherosclerosis.

BACKGROUND: Oxidation of low-density lipoprotein (LDL) and the subsequent processing of oxidized LDL (oxLDL) by macrophages results in activation of specific T cells, which contributes to the development of atherosclerosis. Oral tolerance induction and the subsequent activation of regulatory T cells may be an adequate therapy for the treatment of atherosclerosis. METHODS AND RESULTS: Tolerance to oxLDL and malondialdehyde-treated LDL (MDA-LDL) was induced in LDL receptor-/- mice fed a Western-type diet by oral administration of oxLDL or MDA-LDL before the induction of atherogenesis. Oral tolerance to oxLDL resulted in a significant attenuation of the initiation (30% to 71%; P<0.05) and progression (45%; P<0.05) of atherogenesis. Tolerance to oxLDL induced a significant increase in CD4+ CD25+ Foxp3+ cells in spleen and mesenteric lymph nodes, and these cells specifically responded to oxLDL with increased transforming growth factor-beta production. Tolerance to oxLDL also increased the mRNA expression of Foxp3, CTLA-4, and CD25 in the plaque. In contrast, tolerance to MDA-LDL did not affect atherogenesis. CONCLUSIONS: OxLDL-specific T cells, present in LDL receptor-/- mice and important contributors in the immune response leading to atherosclerotic plaque, can be counteracted by oxLDL-specific CD4+ CD25+ Foxp3+ regulatory T cells activated via oral tolerance induction to oxLDL. We conclude that the induction of oral tolerance to oxLDL may be a promising strategy to modulate the immune response during atherogenesis and a new way to treat atherosclerosis.

Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling.

The eye lens contains a structural protein, alpha crystallin, composed of two homologous primary gene products alpha A2 and alpha B2. In certain rodents, still another alpha crystallin polypeptide, alpha AIns, occurs, which is identical to alpha A2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete alpha A crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the alpha A gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5' non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the alpha AIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3' non-coding region. The second exon is bordered by a 3' splice junction (A X G/G X C), which deviates from the consensus for donor splice sites (A X G/G X T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.

Bovine beta-crystallin complementary DNA clones. Alternating proline/alanine sequence of beta B1 subunit originates from a repetitive DNA sequence.

A library of recombinant plasmids carrying complementary DNA sequences synthesized from bovine lens messenger RNAs was constructed. Clones coding for five different beta-crystallin subunits: beta B1, beta B3, beta Bp, beta s, beta A3 (and beta A1), were identified by means of hybridization selection, followed by one- and two-dimensional gel electrophoresis of the translational products. Under rather stringent conditions each of these clones hybridizes with its corresponding mRNA and does not show significant cross-hybridization with mRNAs coding for other beta-crystallins, except in the case of the homologous beta A3 and beta A1-crystallins. The beta A3 and beta A1 subunits seem to be encoded by one mRNA using two different AUG codons as start position for translation. We have also determined the nucleotide sequence of a beta B1-crystallin cDNA (pBL beta B1) which enabled us to deduce the complete amino acid sequence of the protein. The beta B1-crystallin, a characteristic component of the high molecular weight crystallin aggregate (beta H), is internally homologous both at DNA and protein level as has been reported for gamma- and other beta-crystallins. This is in agreement with the idea that these proteins had a common ancestral precursor gene that internally duplicated. The G + C content of the coding sequence of beta B1 is very high: 67% overall and even 84.2% for the first 170 nucleotides, due to a remarkable non-random codon usage. A proline/alanine repetition in the N-terminal domain of the protein is encoded by a repetitive "simple" DNA sequence.

Flow cytometric characterisation of antigen presenting dendritic cells after in vitro exposure to diesel exhaust particles.

The aim of this study was to obtain more insight into the effect of diesel exhaust particles (DEP) on the maturation of primary human dendritic cells. Monocyte-derived dendritic cells (Mo-DC) derived from seven different donors were exposed to different DEP concentrations (0.2,2,20,200 and 2,000 ng/ml) in the presence or absence of lipopolysaccharide (LPS), and changes in the surface expression of HLA-DR, CD86 and CD83 were examined. Exposure of Mo-DC to DEP alone did not alter expression levels of any of the markers. Treatment with LPS alone increased the expression levels of all three surface markers, although the levels were not significantly different compared to untreated DCs. The LPS-induced marker expression could be further enhanced by co-stimulation of the cells with DEP. Statistical significantly increased levels of CD83 expression were observed after exposure to 0.2 (p=0.018), 20 (p=0.010) and 200 ng/ml (p=0.047) DEP combined with LPS in the group of responders. We conclude that DEP has an adjuvant effect on LPS-induced maturation of Mo-DC.

Expression analysis of immune-related genes in CD34(+) progenitor-derived dendritic cells after exposure to the chemical contact allergen DNCB.

We studied the changes in gene expression after exposure of human dendritic cells (DCs) to the model allergen dinitrochlorobenzene (DNCB). DCs were derived from CD34(+) progenitor cells of three different donors and exposed to 10 microM DNCB or solvent for several time intervals (3, 6 and 12h). cDNA microarrays were used to assess the transcriptional activity of 11,000 human genes. Compared to control gene expression, changes larger than +/-two-fold were observed for 241 genes after exposure to DNCB. Of these genes, 137 were up-regulated and 104 down-regulated. Twenty of these genes encode proteins that are related to the immune response (cytokines, chemokines, their receptors, cytokine/chemokines-related genes, transcription and signal transduction genes) and are discussed in more detail. Our data indicate that exposure to DNCB does not induce a typical maturation pattern in DCs.

Stromal cells in long-term cultures of liver, spleen, and bone marrow at different developmental ages have different capacities to maintain GM-CFC proliferation.

Measurements were made of the granulocyte-macrophage colony-forming cell (GM-CFC) yield in long-term cultures established from different combinations of stroma and hemopoietic recharge inocula derived from hemopoietic organs at different stages of their embryological development. Results indicated differences in the supporting capacity of the stroma, related to the hemopoietic activity of the organ of origin. Stroma derived from hemopoietically active organs (adult bone marrow, neonatal spleen, fetal liver) supported the proliferation of GM-CFC to a larger extent than stroma derived from organs with a low hemopoietic activity (neonatal bone marrow liver at 2 days; spleen at 3 weeks). Regardless of the origin of the hemopoietic cells, stroma from adult bone marrow displayed the highest ability to support GM-CFC proliferation. The capacity of GM-CFC from hemopoietic recharge cell populations to proliferate on stroma was not related to the hemopoietic activity of their organ of origin. Regardless of their organ of origin the GM-CFC present in each of the different hemopoietic recharge populations were able to proliferate provided that they were seeded on an appropriate stroma. These experiments showed that stromal cells cultured from hemopoietic organs at different developmental ages determine the hemopoietic activity of long-term cultures as measured via GM-CFC recovery.

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.

The monoclonal antibodies 18d7/91f2 recognize a receptor regulatory protein on mouse bone marrow stromal cells.

Two monoclonal antibodies 18D7 and 91F2 were developed by immunizing rats with the mouse bone marrow-derived osteogenic cell line MN7. Hybridomas secreting rat antibodies against MN7 cell surface markers were selected by flow cytometry analysis. Both the monoclonal antibody 18D7 and the monoclonal antibody 91F2 are directed against the same cell surface antigen present on MN7 cells. Here, we report on the immunopurification of the 18D7/91F2 antigen and its identification as the prostaglandin F2 alpha receptor regulatory protein (FPRP). FPRP is expressed as a single messenger RNA (mRNA) of approximately 6 kilobases (kb) in MN7 cells and is differentially expressed in developing osteogenic cultures of bone marrow cells of the mouse. However, addition of the monoclonal antibodies 18D7 and 91F2 to these cultures did not inhibit bone formation in vitro. Both monoclonal antibodies reacted with mouse stromal cell lines established from bone marrow, thymus, spleen, and mandibular condyles. Immunohistochemical analysis of mature tibia of mice using the monoclonal antibody 18D7 revealed the presence of a distinct population of bone marrow cells close to trabecular and endosteal bone surfaces. In the central bone marrow, hardly any positive cells were found. In 17-day-old fetal mouse radius 18D7 immunoreactivity was restricted to cells in the periosteum in close vicinity to the bone collar. Mature osteoblasts, osteoclasts, osteocytes, growth plate chondrocytes, and mature macrophages were all negative. Taken together, these results suggest that FPRP plays a role in the osteogenic differentiation process.

Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry.

Well-resolved ion signals of intact large protein assemblies, with molecular masses extending above one million Dalton, have been detected and mass analyzed using electrospray ionization mass spectrometry, with an uncertainty in mass of <0.2%. The mass spectral data seem to reflect known solution-phase behavior of the studied protein assembly and have therefore been directly used to probe the protein assembly topology and stability as a function of ionic strength and pH.

Culture of hematopoietic stem cells purified from murine bone marrow.

The results of the Y-chromosome in situ hybridization experiments, the MRA assessment, and the long-term production of CFU-GM in vitro indicate that our protocol to sort low density WGA+, 15/1.1-, Rh123 dull cells enriches about 200-fold for PHSC. Assays for spleen colony formation (CFU-S) and radioprotection (30-day survival) were shown to be unspecific for PHSC, and, therefore, we lack a quantitative PHSC assay. The absolute number of PHSC in the bone marrow is not known any more, the purity of our sorted population likewise is unknown. Long-term repopulating cells (PHSC) could be separated from short-term repopulating ones by using Rh123 staining. The short-term repopulating cells (Rh123 bright) provided sufficient offspring to protect lethally irradiated mice until endogenous PHSC could reconstitute hematopoiesis. These cells are therefore of interest for bone marrow transplantation, because they provide radioprotection without long-term repopulation and graft-versus-host disease. For gene therapy these cells are of limited use, and PHSC with extensive replication are needed. The PHSC were not cultured successfully. Less than 15% of the sorted Rh123 dull cells responded in semisolid or liquid cultures in the presence of growth factors. Proliferation without differentiation was not observed. This may indicate that the right growth factor has not been found yet. On the other hand, about 30% of the cells responded in stromal layers of long-term bone marrow cultures and prolonged CFU-GM production and cobblestone area formation were observed there, suggesting that cell-cell contact and adherence molecules play a regulatory role in PHSC replication.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges.

Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro, but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 x 10(6) cells are loaded, mineralization as measured by 85Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10(7) cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phosphatase positive cells. However; synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.

Direction of the reactivity of vanillyl-alcohol oxidase with 4-alkylphenols.

The covalent flavoprotein vanillyl-alcohol oxidase (VAO) predominantly converts short-chain 4-alkylphenols, like 4-ethylphenol, to (R)-1-(4'-hydroxyphenyl)alcohols and medium-chain 4-alkylphenols, like 4-butylphenol, to 1-(4'-hydroxyphenyl)alkenes. Crystallographic studies have indicated that the active site residue Asp170 is involved in determining the efficiency of substrate hydroxylation. To test this hypothesis, we have addressed the reactivity of Asp170 variants with 4-alkylphenols. The substrate preference of Asp170Glu was similar to wild type VAO. However, Asp170Ser was most active with branched-chain 4-alkylphenols. The hydroxylation efficiency of the Asp170 variants was dependent on the bulkiness of the newly introduced side chain. The Glu170 mutation favored the production of alkenes, whereas the Ser170 mutation stimulated the formation of alcohols.

Tuning of the product spectrum of vanillyl-alcohol oxidase by medium engineering.

The flavoenzyme vanillyl-alcohol oxidase (VAO) catalyzes the conversion of 4-alkylphenols through the initial formation of p-quinone methide intermediates. These electrophilic species are stereospecifically attacked by water to yield (R)-1-(4'-hydroxyphenyl)alcohols or rearranged in a competing reaction to 1-(4'-hydroxyphenyl)alkenes. Here, we show that the product spectrum of VAO can be controlled by medium engineering. When the enzymatic conversion of 4-propylphenol was performed in organic solvent, the concentration of the alcohol decreased and the concentration of the cis-alkene, but not the trans-alkene, increased. This change in selectivity occurred in both toluene and acetonitrile and was dependent on the water activity of the reaction medium. A similar shift in alcohol/cis-alkene product ratio was observed when the VAO-mediated conversion of 4-propylphenol was performed in the presence of monovalent anions that bind specifically near the enzyme active site.

The effect of age on the systemic absorption, disposition and pharmacodynamics of bupivacaine after epidural administration.

The influence of age on the systemic absorption and disposition of bupivacaine following epidural administration in 20 male patients (22 to 81 years) was examined using a stable isotope method to determine whether pharmacokinetics play a role in age-related pharmacodynamic changes seen with the drug. After epidural bupivacaine administration a deuterium-labelled analogue was administered intravenously. Bi- and triexponential functions were fitted to plasma concentration-time data of deuterium-labelled bupivacaine. The systemic absorption was described by 2 parallel first-order absorption processes. The upper level of analgesia and the duration of analgesia at dermatome T-12 increased with age (r = 0.68, p less than 0.001; r = 0.56, p less than 0.01, respectively). The time to maximal caudad spread of analgesia and the time to onset of motor block decreased with age (r = -0.76, p less than 0.0001; r = -0.72, p less than 0.001, respectively). Age did not influence systemic absorption or disposition of bupivacaine. We conclude that the changes in the clinical profile of bupivacaine with age are not due to altered pharmacokinetics, but may be related to changes in the pharmacodynamics of the drug.

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice.

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.