Alterations in high-density lipoprotein proteome and function associated with persistent organic pollutants.

There is a growing body of evidence that persistent organic pollutants (POPs) may increase the risk for cardiovascular disease (CVD), but the mechanisms remain unclear. High-density lipoprotein (HDL) acts protective against CVD by different processes, and we have earlier found that HDL from subjects with CVD contains higher levels of POPs than healthy controls. In the present study, we have expanded analyses on the same individuals living in a contaminated community and investigated the relationship between the HDL POP levels and protein composition/function. HDL from 17 subjects was isolated by ultracentrifugation. HDL protein composition, using nanoliquid chromatography tandem mass spectrometry, and antioxidant activity were analyzed. The associations of 16 POPs, including polychlorinated biphenyls (PCBs) and organochlorine pesticides, with HDL proteins/functions were investigated by partial least square and multiple linear regression analysis. Proteomic analyses identified 118 HDL proteins, of which ten were significantly (p<0.05) and positively associated with the combined level of POPs or with highly chlorinated PCB congeners. Among these, cholesteryl ester transfer protein and phospholipid transfer protein, as well as the inflammatory marker serum amyloid A, were found. The serum paraoxonase/arylesterase 1 activity was inversely associated with POPs. Pathway analysis demonstrated that up-regulated proteins were associated with biological processes involving lipoprotein metabolism, while down-regulated proteins were associated with processes such as negative regulation of proteinases, acute phase response, platelet degranulation, and complement activation. These results indicate an association between POP levels, especially highly chlorinated PCBs, and HDL protein alterations that may result in a less functional particle. Further studies are needed to determine causality and the importance of other environmental factors. Nevertheless, this study provides a first insight into a possible link between exposure to POPs and risk of CVD.

Distinctive proteomic profiles among different regions of human carotid plaques in men and women.

The heterogeneity of atherosclerotic tissue has limited comprehension in proteomic and metabolomic analyses. To elucidate the functional implications, and differences between genders, of atherosclerotic lesion formation we investigated protein profiles from different regions of human carotid atherosclerotic arteries; internal control, fatty streak, plaque shoulder, plaque centre, and fibrous cap. Proteomic analysis was performed using 2-DE with MALDI-TOF, with validation using nLC-MS/MS. Protein mapping of 2-DE identified 52 unique proteins, including 15 previously unmapped proteins, of which 41 proteins were confirmed by nLC-MS/MS analysis. Expression levels of 18 proteins were significantly altered in plaque regions compared to the internal control region. Nine proteins showed site-specific alterations, irrespective of gender, with clear associations to extracellular matrix remodelling. Five proteins display gender-specific alterations with 2-DE, with two alterations validated by nLC-MS/MS. Gender differences in ferritin light chain and transthyretin were validated using both techniques. Validation of immunohistochemistry confirmed significantly higher levels of ferritin in plaques from male patients. Proteomic analysis of different plaque regions has reduced the effects of plaque heterogeneity, and significant differences in protein expression are determined in specific regions and between genders. These proteomes have functional implications in plaque progression and are of importance in understanding gender differences in atherosclerosis.

Lipoprotein profiles in human heterozygote carriers of a functional mutation P297S in scavenger receptor class B1.

The scavenger receptor class B type 1 (SR-B1) is an important HDL receptor involved in cholesterol uptake and efflux, but its physiological role in human lipoprotein metabolism is not fully understood. Heterozygous carriers of the SR-B1(P297S) mutation are characterized by increased HDL cholesterol levels, impaired cholesterol efflux from macrophages and attenuated adrenal function. Here, the composition and function of lipoproteins were studied in SR-B1(P297S) heterozygotes.Lipoproteins from six SR-B1(P297S) carriers and six family controls were investigated. HDL and LDL/VLDL were isolated by ultracentrifugation and proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. HDL antioxidant properties, paraoxonase 1 activities, apoA-I methionine oxidations and HDL cholesterol efflux capacity were assessed.Multivariate modeling separated carriers from controls based on lipoprotein composition. Protein analyses showed a significant enrichment of apoE in LDL/VLDL and of apoL-1 in HDL from heterozygotes compared to controls. The relative distribution of plasma apoE was increased in LDL and in lipid-free form. There were no significant differences in paraoxonase 1 activities, HDL antioxidant properties or HDL cholesterol efflux capacity but heterozygotes showed a significant increase of oxidized methionines in apoA-I.The SR-B1(P297S) mutation affects both HDL and LDL/VLDL protein compositions. The increase of apoE in carriers suggests a compensatory mechanism for attenuated SR-B1 mediated cholesterol uptake by HDL. Increased methionine oxidation may affect HDL function by reducing apoA-I binding to its targets. The results illustrate the complexity of lipoprotein metabolism that has to be taken into account in future therapeutic strategies aiming at targeting SR-B1.

Structure of HDL: particle subclasses and molecular components.

A molecular understanding of high-density lipoprotein (HDL) will allow a more complete grasp of its interactions with key plasma remodelling factors and with cell-surface proteins that mediate HDL assembly and clearance. However, these particles are notoriously heterogeneous in terms of almost every physical, chemical and biological property. Furthermore, HDL particles have not lent themselves to high-resolution structural study through mainstream techniques like nuclear magnetic resonance and X-ray crystallography; investigators have therefore had to use a series of lower resolution methods to derive a general structural understanding of these enigmatic particles. This chapter reviews current knowledge of the composition, structure and heterogeneity of human plasma HDL. The multifaceted composition of the HDL proteome, the multiple major protein isoforms involving translational and posttranslational modifications, the rapidly expanding knowledge of the HDL lipidome, the highly complex world of HDL subclasses and putative models of HDL particle structure are extensively discussed. A brief history of structural studies of both plasma-derived and recombinant forms of HDL is presented with a focus on detailed structural models that have been derived from a range of techniques spanning mass spectrometry to molecular dynamics.

Persistent organic pollutants distribution in lipoprotein fractions in relation to cardiovascular disease and cancer.

Persistent organic pollutants (POPs) are lipophilic environmental toxins that have been associated with cardiovascular disease (CVD) and cancer. The aim of this study was to investigate the concentrations of POPs in human high and low/very low-density lipoproteins (HDL and LDL/VLDL) and the possible association with CVD and cancer occurrence in individuals living in a contaminated area. Lipoproteins from 28 individuals (7 healthy controls, 8 subjects with cancer, 13 subjects with CVD) were isolated and the fraction-specific concentration of 20 different POPs was analyzed by high resolution gas chromatography/high resolution mass spectrometry. The activity of Paraoxonase 1 (PON1), an anti-oxidant in HDL, was determined in plasma of these 28 subjects and additional 50 subjects from the same area excluding diseases other than cancer or CVD. Fourteen polychlorinated biphenyls (PCBs) and three organochlorine pesticides were detected, and especially highly chlorinated PCBs were enriched in lipoproteins. Significantly higher concentrations of POPs were found among individuals with CVD or cancer compared to controls. Principal component analyses showed that POP concentrations in HDL were more associated with CVD, while POP concentrations in LDL/VLDL were more associated with cancer. PON1 activity was negatively correlated to sumPCB and a co-variation between decreased arylesterase-activity, increased PCB concentrations and CVD was found. This study shows that POPs are present in lipoproteins and were more abundant in individuals with CVD or cancer compared to healthy controls. The results also indicate that PCB exposure is accompanied by reduced PON1 activity that could impair the HDL function to protect against oxidation.

ApoA-I mutations, L202P and K131del, in HDL from heterozygotes with low HDL-C.

PURPOSE: Mutations in apolipoprotein A-I (apoA-I) may affect plasma high-density lipoprotein (HDL) cholesterol levels and the risk for cardiovascular disease but little is known about the presence and effects of circulating apoA-I variants. This study investigates whether the apoA-I mutations, apoA-I(L202P) and apoA-I(K131del) , are present on plasma HDL particles derived from heterozygote carriers and whether this is associated to changes in HDL protein composition. EXPERIMENTAL DESIGN: Plasma HDL of heterozygotes for either apoA-I(L202P) or apoA-I(K131del) and family controls was isolated using ultracentrifugation. HDL proteins were separated by 2DE and analyzed by MS. RESULTS: ApoA-I peptides containing apoA-I(L202P) or apoA-I(K131del) were identified in HDL from heterozygotes. The apoA-I(L202P) mutant peptide was less abundant than wild-type peptide while the apoA-I(K131del) mutant peptide was more abundant than wild-type peptide in the heterozygotes. Two-dimensional gel electrophoresis analyses indicated that, compared to controls, HDL in apoA-I(L202P) carriers contained less apoE and more zinc-α-2-glycoprotein while HDL from the apoA-I(K131del) heterozygotes contained more alpha-1-antitrypsin and transthyretin. CONCLUSIONS AND CLINICAL RELEVANCE: Both apoA-I(L202P) and apoA-I(K131del) were identified in HDL. In heterozygotes, these mutations have markedly differential effects on the concentration of wild-type apoA-I in the circulation, as well as the HDL proteome, both of which might affect the clinical phenotype encountered in the heterozygous carriers.

Airway symptoms and biological markers in nasal lavage fluid in subjects exposed to metalworking fluids.

BACKGROUNDS: Occurrence of airway irritation among industrial metal workers was investigated. The aims were to study the association between exposures from water-based metal working fluids (MWF) and the health outcome among the personnel, to assess potential effects on the proteome in nasal mucous membranes, and evaluate preventive actions. METHODS: The prevalence of airway symptoms related to work were examined among 271 metalworkers exposed to MWF and 24 metal workers not exposed to MWF at the same factory. At the same time, air levels of potentially harmful substances (oil mist, morpholine, monoethanolamine, formaldehyde) generated from MWF was measured. Nasal lavage fluid was collected from 13 workers and 15 controls and protein profiles were determined by a proteomic approach. RESULTS: Airway symptoms were reported in 39% of the workers exposed to MWF although the measured levels of MWF substances in the work place air were low. Highest prevalence was found among workers handling the MWF machines but also those working in the same hall were affected. Improvement of the ventilation to reduce MWF exposure lowered the prevalence of airway problems. Protein profiling showed significantly higher levels of S100-A9 and lower levels of SPLUNC1, cystatin SN, Ig J and ß2-microglobulin among workers with airway symptoms. CONCLUSIONS: This study confirms that upper airway symptoms among metal workers are a common problem and despite low levels of MWF-generated substances, effects on airway immune proteins are found. Further studies to clarify the role of specific MWF components in connection to airway inflammation and the identified biological markers are warranted.

Airway irritation among indoor swimming pool personnel: trichloramine exposure, exhaled NO and protein profiling of nasal lavage fluids.

PURPOSE: Occurrence of airway irritation among indoor swimming pool personnel was investigated. The aims of this study were to assess trichloramine exposure levels and exhaled nitric oxide in relation to the prevalence of airway symptoms in swimming pool facilities and to determine protein effects in the upper respiratory tract. METHODS: The presence of airway symptoms related to work was examined in 146 individuals working at 46 indoor swimming pool facilities. Levels of trichloramine, as well as exhaled nitric oxide, were measured in five facilities with high prevalence of airway irritation and four facilities with no airway irritation among the personnel. Nasal lavage fluid was collected, and protein profiles were determined by a proteomic approach. RESULTS: 17 % of the swimming pool personnel reported airway symptoms related to work. The levels of trichloramine in the swimming pool facilities ranged from 0.04 to 0.36 mg/m(3). There was no covariance between trichloramine levels, exhaled nitric oxide and prevalence of airway symptoms. Protein profiling of the nasal lavage fluid showed that the levels alpha-1-antitrypsin and lactoferrin were significantly higher, and S100-A8 was significantly lower in swimming pool personnel. CONCLUSIONS: This study confirms the occurrence of airway irritation among indoor swimming pool personnel. Our results indicate altered levels of innate immunity proteins in the upper airways that may pose as potential biomarkers. However, swimming pool facilities with high prevalence of airway irritation could not be explained by higher trichloramine exposure levels. Further studies are needed to clarify the environmental factors in indoor swimming pools that cause airway problems and affect the immune system.

Heterozygosity for a loss-of-function mutation in GALNT2 improves plasma triglyceride clearance in man.

Genome-wide association studies have identified GALNT2 as a candidate gene in lipid metabolism, but it is not known how the encoded enzyme ppGalNAc-T2, which contributes to the initiation of mucin-type O-linked glycosylation, mediates this effect. In two probands with elevated plasma high-density lipoprotein cholesterol and reduced triglycerides, we identified a mutation in GALNT2. It is shown that carriers have improved postprandial triglyceride clearance, which is likely attributable to attenuated glycosylation of apolipoprotein (apo) C-III, as observed in their plasma. This protein inhibits lipoprotein lipase (LPL), which hydrolyses plasma triglycerides. We show that an apoC-III-based peptide is a substrate for ppGalNAc-T2 while its glycosylation by the mutant enzyme is impaired. In addition, neuraminidase treatment of apoC-III which removes the sialic acids from its glycan chain decreases its potential to inhibit LPL. Combined, these data suggest that ppGalNAc-T2 can affect lipid metabolism through apoC-III glycosylation, thereby establishing GALNT2 as a lipid-modifying gene.

Innate immunity proteins and a new truncated form of SPLUNC1 in nasopharyngeal aspirates from infants with respiratory syncytial virus infection.

PURPOSE: Respiratory syncytial virus (RSV) is the most common cause of severe respiratory tract infection in infants. The aim was to identify host defence components in nasopharyngeal aspirate (NPA) from infants with RSV infection and to study the expression of the novel 25 kDa innate immunity protein SPLUNC1. EXPERIMENTAL DESIGN: NPAs from infants were analyzed with 2-DE and MS in a pilot study. The levels of SPLUNC1 were analyzed with immunoblotting in 47 NPAs, admitted for RSV diagnosis. RESULTS: Totally, 35 proteins were identified in NPA, including several innate immunity proteins such as group X phospholipase A(2) , different S100 proteins and SPLUNC1. In addition, a new truncated 15 kDa form of SPLUNC1 was identified that was detected in about 50% of the aspirates admitted for RSV diagnosis. RSV-positive boys had significantly less 25 kDa SPLUNC1 than RSV-negative boys while there were no significant differences among girls. CONCLUSIONS AND CLINICAL RELEVANCE: Several important innate immunity proteins were identified in NPA. Notably, a new truncated form of the newly suggested anti-bacterial protein SPLUNC1 was found. It is possible that a decrease in SPLUNC1 in the upper airways may increase the risk for severe pneumonia in boys.

High-density lipoprotein proteome dynamics in human endotoxemia.

BACKGROUND: A large variety of proteins involved in inflammation, coagulation, lipid-oxidation and lipid metabolism have been associated with high-density lipoprotein (HDL) and it is anticipated that changes in the HDL proteome have implications for the multiple functions of HDL. Here, SELDI-TOF mass spectrometry (MS) was used to study the dynamic changes of HDL protein composition in a human experimental low-dose endotoxemia model. Ten healthy men with low HDL cholesterol (0.7+/-0.1 mmol/L) and 10 men with high HDL cholesterol levels (1.9+/-0.4 mmol/L) were challenged with endotoxin (LPS) intravenously (1 ng/kg bodyweight). We previously showed that subjects with low HDL cholesterol are more susceptible to an inflammatory challenge. The current study tested the hypothesis that this discrepancy may be related to differences in the HDL proteome. RESULTS: Plasma drawn at 7 time-points over a 24 hour time period after LPS challenge was used for direct capture of HDL using antibodies against apolipoprotein A-I followed by subsequent SELDI-TOF MS profiling. Upon LPS administration, profound changes in 21 markers (adjusted p-value < 0.05) were observed in the proteome in both study groups. These changes were observed 1 hour after LPS infusion and sustained up to 24 hours, but unexpectedly were not different between the 2 study groups. Hierarchical clustering of the protein spectra at all time points of all individuals revealed 3 distinct clusters, which were largely independent of baseline HDL cholesterol levels but correlated with paraoxonase 1 activity. The acute phase protein serum amyloid A-1/2 (SAA-1/2) was clearly upregulated after LPS infusion in both groups and comprised both native and N-terminal truncated variants that were identified by two-dimensional gel electrophoresis and mass spectrometry. Individuals of one of the clusters were distinguished by a lower SAA-1/2 response after LPS challenge and a delayed time-response of the truncated variants. CONCLUSIONS: This study shows that the semi-quantitative differences in the HDL proteome as assessed by SELDI-TOF MS cannot explain why subjects with low HDL cholesterol are more susceptible to a challenge with LPS than those with high HDL cholesterol. Instead the results indicate that hierarchical clustering could be useful to predict HDL functionality in acute phase responses towards LPS.

Wear particles from studded tires and granite pavement induce pro-inflammatory alterations in human monocyte-derived macrophages: a proteomic study.

Airborne particulate matter is considered to be one of the environmental contributors to the mortality in cancer, respiratory, and cardiovascular diseases. For future preventive actions, it is of major concern to investigate the toxicity of defined groups of airborne particles and to clarify their pathways in biological tissues. To expand the knowledge beyond general inflammatory markers, this study examined the toxicoproteomic effects on human monocyte derived macrophages after exposure to wear particles generated from the interface of studded tires and a granite-containing pavement. As comparison, the effect of endotoxin was also investigated. The macrophage proteome was separated using two-dimensional gel electrophoresis. Detected proteins were quantified, and selected proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry. Among analyzed proteins, seven were significantly decreased and three were increased by exposure to wear particles as compared to unexposed control cells. Endotoxin exposure resulted in significant changes in the expression of six proteins: four decreased and two increased. For example, macrophage capping protein was significantly increased after wear particle exposure only, whereas calgizzarin and galectin-3 were increased by both wear particle and endotoxin exposure. Overall, proteins associated with inflammatory response were increased and proteins involved in cellular functions such as redox balance, anti-inflammatory response, and glycolysis were decreased. Investigating the effects of characterized wear particles on human macrophages with a toxicoproteomic approach has shown to be useful in the search for more detailed information about specific pathways and possible biological markers.

Protein profiling of low-density lipoprotein from obese subjects.

Although obesity and high levels of low-density lipoprotein (LDL) are well-known risk factors for cardiovascular disease, the precise role(s) of different LDL constituents in obesity has not been explored. In the present study, we compared the LDL proteome of healthy control adults (body mass index<25) and obese subjects (body mass index>30). LDL was isolated by density-gradient ultracentrifugation and proteins were separated with 2-D PAGE, quantified, and identified by peptide mass fingerprinting using MALDI-TOF MS. A new LDL-associated protein was identified as transthyretin and found to be significantly more abundant in LDL from the obese subjects. In addition, LDL from the obese subjects contained relatively more α(1) -antitrypsin, apo J, apo C-II, than LDL from controls, and also more of an acidic isoform (pI/Mr; 5.2/23 100) of apo A-I. On the other hand, the relative amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3/23 100) were significantly less in LDL from the obese subjects. Apo E was less and non-sialylated apo C-III more abundant in LDL from obese men than control men, while there were no such differences between LDL from obese and control women. These findings illustrate that obesity is not only associated with increased LDL-cholesterol levels but also with alterations in the LDL protein composition. The presence of transthyretin in LDL from obese subjects may reflect over-nutrition and affect the lipid metabolism in obesity.

Characterization of apolipoprotein M isoforms in low-density lipoprotein.

Apo M is a recently discovered human lipoprotein thought to be involved in the metabolism of lipids and lipoprotein particles. Here, a proteomic approach was applied to examine the glycosylation pattern of apo M in human LDL. We treated LDL proteins with N-glycosidase or neuraminidase, studied mobility shifts of Apo M by two-dimensional gel electrophoresis, and different isoforms were then identified with mass spectrometry. This way, we demonstrated the presence of five isoforms of apo M in LDL: three that are both N-glycosylated and sialylated, one that is N-glycosylated but not sialylated, and one that is neither N-glycosylated nor sialylated. As judged from the examination of LDL from 20 healthy human subjects, the three N-glycosylated and sialylated forms are most abundant (80-100% of the total apo M in LDL) whereas the unsialylated and unglycosylated variants constitute at most 20%. Comparative analysis showed that the same five isoforms of apo M are also present in HDL. Further studies aiming at elucidating the role of apo M in health and disease will have to take this polymorphism of apo M proteins into account.

Test performance and self-esteem in relation to experienced stress in Swedish sixth and ninth graders--saliva cortisol levels and psychological reactions to demands.

AIMS: To study relations between test performance, academic self-esteem, self-reported stress and saliva cortisol levels in students exposed to test demands at school. METHODS: 46 randomly selected 6th and 9th graders voluntarily participated in an experimental school test concerning reading and mathematics skills. Cortisol saliva samples were collected at awakening, 30 min later, before test and after test. Perceived stress was registered with a visual analogue scale (VAS). A standardized self-rating questionnaire for measuring self-esteem was administered together with questions about school stress and psychological reactions when exposed to stress during a test. RESULTS: Experienced stress during the school test was correlated to low test performance and to low self-rated evaluation of self-esteem as well as to an increase of saliva cortisol levels during the test. There were also correlations between certain psychological reactions to demands and different cortisol measures. Children who reported that they applied the procedure "I say to myself: I can solve this task" in a school test situation had a lower morning increase of saliva cortisol. Reported use of the procedure "I get worried and will have problems solving other tasks too"--when referring to a school test situation--was correlated to an increase in cortisol levels during the test situation. CONCLUSION: Test performance, academic self-esteem, perceived stress and reactions of the hypothalamus-pituitary-adrenal axis (HPA axis) are highly intertwined. Furthermore, certain school stress psychological reactions to performance demands seem to be related to different cortisol reactions.

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.

Lipoproteomics I: mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.

The molecular mechanisms underlying the relationship between low-density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information about the protein composition of LDL may contribute to reveal its role in atherogenesis and the mechanisms that lead to coronary disease in humans. Here, we sought to map the proteins in human LDL by a proteomic approach. LDL was isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix assisted laser desorption/ionization-time of flight-mass spectrometry and with amino acid sequencing using electrospray ionization tandem mass spectrometry. These procedures identified apo B-100, apo C-II, apo C-III (three isoforms), apo E (four isoforms), apo A-I (two isoforms), apo A-IV, apo J and apo M (three isoforms not previously described). In addition, three proteins that have not previously been identified in LDL were found: serum amyloid A-IV (two isoforms), calgranulin A, and lysozyme C. The identities of apo M, calgranulin A, and lysozyme C were confirmed by sequence information obtained after collision-induced dissociation fragmentation of peptides characteristic for these proteins. Moreover, the presence of lysozyme C was further corroborated by demonstrating enriched hydrolytic activity in LDL against Micrococcus lysodeikticus. These results indicate that in addition to the dominating apo B-100, LDL contains a number of other apolipoproteins, many of which occur in different isoforms. The demonstration, for the first time, that LDL contains calgranulin A and lysozyme C raises the possibility that LDL proteins may play hitherto unknown role(s) in immune and inflammatory reactions of the arterial wall.

Phospholipase A2 mRNA expression in the nasal mucosa of healthy subjects and patients with seasonal allergic rhinitis.

Phospholipase A2 (PLA2) is a family of enzymes that play different role(s) in inflammation, but their importance in seasonal allergic rhinitis (SAR) has not been clarified. Here, we determined the levels of messenger ribonucleic acid (mRNA) for different PLA2 types in the nasal mucosa of SAR patients (n = 6) and healthy controls (n = 5). Nasal brush samples were taken both during pollen season, when the symptoms of the patients were severe, and off-season, when the patients were free of symptoms. We found that PLA2 IB, IIA, IID, IIE, IIF, III, IVA, IVB, IVC, VIA, VIB, VIIA, VIIB, VIIIA, VIIIB, X, XII and XIII were all expressed in each subject at both occasions. The mRNA levels of PLA2 VIIA (platelet-activating factor (PAF) acetylhydrolase) were lower in SAR patients than controls, both during pollen season (p = 0.03) and off season (p = 0.03). These findings demonstrate that a large number of PLA2 types are expressed in the nasal mucosa, regardless of whether there is ongoing allergic inflammation or not. The observation that PAF acetylhydrolase mRNA expression in the nasal mucosa is lower in SAR patients than in healthy subjects suggests the possibility that impaired ability to inactivate PAF might be of importance in SAR. Further studies are required to clarify whether the decreased PAF acetylhydrolase mRNA expression in SAR is accompanied by decreased enzyme activity and whether aberrations in PAF acetylhydrolase are present in infectious rhinitis patients as well.

Nasal lavage fluid and proteomics as means to identify the effects of the irritating epoxy chemical dimethylbenzylamine.

The aims of this study were to describe the changes in the nasal lavage fluid (NLF) protein pattern after exposure to the irritating epoxy chemical dimethylbenzylamine (DMBA) and to identify the affected proteins using a proteomic approach. The protein patterns of NLF from six healthy subjects and eight epoxy workers with airway irritation were analysed using two-dimensional gel electrophoresis (2-DE) before and after exposure to 100 microg m(-3) DMBA for 2 h in an exposure chamber. NLF proteins were identified by (i) comparison with a 2-DE NLF reference database; (ii) N-terminal amino acid sequencing; and (iii) mass spectrometry. In NLF from healthy subjects, the levels of immunoglobulin A increased and the levels of Clara cell protein 16 (CC16) decreased after chamber exposure, while in NLF from epoxy workers, alpha(2)-macroglobulin and caeruloplasmin increased. Two previously unidentified proteins decreased in NLF from epoxy workers after exposure; these were identified as statherin and calgranulin B. In addition, the subjects who developed high counts of eosinophils in their nasal mucosa after chamber exposure had significantly lower levels of immunoglobulin-binding factor (IgBF) before exposure than subjects with low eosinophil infiltration. These results show that short-term exposure to DMBA causes distinct changes in NLF proteins. Moreover, three proteins that have previously not been associated with upper airway irritation were identified: statherin, calgranulin B and IgBF. Further studies are needed to investigate whether these proteins may be used as biomarkers of airway irritation and to give new insight into the ways in which occupational exposure to irritants causes inflammation of the airways.