Protein In Nasal Lavage Fluid Research Articles

Comorbidity has no impact on eosinophil inflammation in the upper airways or on severity of the sinonasal disease in patients with nasal polyps.

The study was designed to determine whether there is an association between the comorbidity as atopy, bronchial asthma, aspirin intolerance and eosinophil infiltration of the upper airways, severity of the sinonasal disease and rate of revision sinus surgery in patients with nasal polyps. One hundred and fifty patients were enrolled in the prospective study. Differences in CT score, rate of revision surgery, concentration of eotaxin and eosinophil cationic protein in nasal lavage fluid (NALF) and distribution of eosinophils in NALF and nasal tissue in patients with chronic rhinosinusitis with nasal polyps (CRSwNP), chronic rhinosinusitis without nasal polyps (CRSsNP) and control group were investigated. We focused on the relationship between presence of comorbidity (atopy, bronchial asthma and aspirin intolerance) and severity of the disease, the need of revision surgery and markers of eosinophil inflammation in upper airways in patients with CRSwNP. Patients with CRSwNP had more severe form of the sinonasal disease, higher rate of revision FESS and significant higher presence of markers of eosinophil inflammation in NALF and nasal tissue than patients with CRSsNP (P<0.05). Atopic and non-atopic asthma as wellas aspirin sensitivity significantly more often coexisted with CRSwNP. Comorbidity did not influence eosinophil infiltration or severity of the disease in patients with CRSwNP. Presence of comorbidity (atopy, bronchial asthma and aspirin intolerance) has no impact on severity of the disease or eosinophil content in the upper airways in patients with CRSwNP.

Improved preparation of nasal lavage fluid (NLF) as a noninvasive sample for proteomic biomarker discovery

Nasal lavage fluid (NLF) becomes more and more important as a noninvasive patient sample serving as a new opportunity to discover new biomarkers in diverse human diseases comprising mainly respiratory disorders. This was supported by the observation that the protein profile of NLF differs from conventional samples of i.e. whole blood, hence being capable to complement or even expand the so far biomarker index. Since sample acquisition and processing are the most crucial steps for a profound and sensitive identification we present here a modified protocol of NLF generation and measurement. We show that mild washing steps for sample generation followed by column-mediated concentration and acetone precipitation are appropriate steps to minimize serum leakage by concomitantly highlighting proteins which represent typical components of NLF. This is shown by separation of proteins via 2D-PAGE followed by LC-MS/MS as well as Gel-LC-MS/MS measurements of cut and digested protein spots/bands. SignificanceFor a better understanding of the molecular mechanisms underlying respiratory diseases NLF samples are favored sources for protein research. NLF acquisition and sample processing were impaired so far by the problem of blood serum leakage and high salt content. Here, we present a modified protocol of NLF generation leading to the display of typical inventory of NLF proteins combined with reduced salt and serum contaminants. By this, both assay reproducibility and the detection of up- or down-regulated proteins reliably can be discovered in the case of biomarker screenings in a disease versus control design. This article is part of a Special Issue entitled: Neuroproteomics: Applications in Neuroscience and Neurology.

Screening Method Using Selected Reaction Monitoring for Targeted Proteomics Studies of Nasal Lavage Fluid

Proteomic-based studies of nasal lavage fluid (NLF) may identify molecular pathways associated with disease pathology and new biomarker candidates of upper airway diseases. However, most studies have used rather tedious untargeted MS techniques. Selected reaction monitoring (SRM) is a sensitive and specific technique that can be used with high throughput. In this study, we developed a semiquantitative SRM-based method targeting 244 NLF proteins. The protein set was identified through a literature study in combination with untargeted LC-MS/MS analyses of trypsin-digested NLF samples. The SRM assays were designed using MS/MS data either downloaded from a proteomic data repository or experimentally obtained. Each protein is represented by one to five peptides, resulting in 708 SRM assays. Three to four transitions per assay were used to ensure analyte specificity. The majority (69%) of the assays showed good within-day precision (coefficient of variation ≤ 20%). The accuracy of the method was evaluated by analyzing four samples prepared with varying amounts of four proteins. Peptide and protein ratios were in good agreement with expected ratios. In conclusion, a high throughput screening method for relative quantification of 244 NLF proteins was developed. The method should be of general use in any proteomic study of the upper airways.

Clara cell protein in nasal lavage fluid and nasal nitric oxide - biomarkers with anti-inflammatory properties in allergic rhinitis

BackgroundClara cell protein (CC16) is ascribed a protective and anti-inflammatory role in airway inflammation. Lower levels have been observed in asthmatic subjects as well as in subjects with intermittent allergic rhinitis than in healthy controls. Nasal nitric oxide (nNO) is present in high concentrations in the upper airways, and considered a biomarker with beneficial effects, due to inhibition of bacteria and viruses along with stimulation of ciliary motility. The aim of this study was to evaluate the presumed anti-inflammatory effects of nasal CC16 and nNO in subjects with allergic rhinitis.MethodsThe levels of CC16 in nasal lavage fluids, achieved from subjects with persistent allergic rhinitis (n = 13), intermittent allergic rhinitis in an allergen free interval (n = 5) and healthy controls (n = 7), were analyzed by Western blot. The levels of nNO were measured by the subtraction method using NIOX®. The occurrences of effector cells in allergic inflammation, i.e. metachromatic cells (MC, mast cells and basophiles) and eosinophils (Eos) were analyzed by light microscopy in samples achieved by nasal brushing.ResultsThe levels of CC16 correlated with nNO levels (r2 = 0.37; p = 0.02) in allergic subjects.The levels of both biomarkers showed inverse relationships with MC occurrence, as higher levels of CC16 (p = 0.03) and nNO (p = 0.05) were found in allergic subjects with no demonstrable MC compared to the levels in subjects with demonstrable MC. Similar relationships, but not reaching significance, were observed between the CC16 and nNO levels and Eos occurrence. The levels of CC16 and nNO did not differ between the allergic and the control groups.ConclusionsThe correlation between nasal CC16 and nNO levels in patients with allergic rhinitis, along with an inverse relationship between their levels and the occurrences of MC in allergic inflammation, may indicate that both biomarkers have anti-inflammatory effects by suppression of cell recruitment. The mechanisms behind these observations warrant further analyses.

Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis

Decreased epithelial expression of mRNA for S100A7 (psoriasin) and S100A8/A9 (calprotectin) has been reported in patients with chronic rhinosinusitis (CRS). We sought to assess whether the expression of S100 proteins is also altered in the sinonasal cavity of patients with CRS. We determined levels of S100 proteins in nasal lavage fluid and sinonasal tissue extracts from patients with CRS using ELISA and immunohistochemical analysis of nasal polyp tissue from patients with CRS with nasal polyps and uncinate tissue from healthy control subjects, patients with CRSsNP, and patients with CRSwNP. Expression levels of S100 proteins were decreased compared with those seen in control subjects in nasal lavage fluid from both CRS groups (P < .05). Similarly, tissue expression of these proteins assessed by means of immunohistochemistry demonstrated clear reductions, primarily in the epithelial lining. Interestingly, levels of calprotectin were increased in nasal polyp tissue despite lower levels in lavage fluid. Levels of calprotectin in nasal tissues were correlated with levels of neutrophils, as assessed by means of quantification of neutrophil elastase. Several S100 proteins are in the epidermal differentiation complex of genes and have been demonstrated to play a role in maintenance of barrier function and formation of an antimicrobial shield. We demonstrate significantly decreased levels of expression of S100 proteins in the epithelium of patients with CRS, which might lead to diminished innate immune responses and barrier function. Increased levels of calprotectin in nasal polyp tissue might reflect neutrophil recruitment and a compensatory mechanism. Future studies will be important to determine whether reduced levels of S100 proteins lead to decreased antimicrobial responses in the upper airways and sinuses and whether this reduction plays a causative role in CRS pathogenesis and susceptibility to infectious disease.

Extensive fractionation and identification of proteins within nasal lavage fluids from allergic rhinitis and asthmatic chronic rhinosinusitis patients

Allergic rhinitis (AR), chronic rhinosinusitis (CRS), and asthma are prevalent airway diseases that can have a substantial impact on a patient's quality of life. MS analyses of biological fluids can effectively screen for proteins associated with disease processes, however, initial detection of diagnostic proteins is difficult due to protein complexity and dynamic range. To enhance the detection of lower abundance proteins, intact nasal lavage fluid (NLF) proteins from nonpolypoid AR and from asthmatic CRS patients were extensively fractionated prior to LC/MS/MS analysis. Pooled NLF samples were processed to remove low molecular weight molecules and high abundance plasma proteins. Anion exchange (AX) chromatography followed by RP-LC further separated the remaining intact NLF proteins. The resulting fractions were digested with trypsin and the peptides analyzed by LC/MS/MS. Spectra were searched with MASCOT, SEQUEST, and X!Tandem to obtain peptide identifications and subsequently analyzed by Scaffold software to identify parent proteins with at least 99% confidence. The 197 identified proteins are compared to those previously cited in the literature and the workflow evaluated to determine the usefulness for the detection of lower abundance proteins. This is the first extensive list of NLF proteins generated from CRS patients with coexisting asthma.

Evaluation of nasal IgA secretion in normal subjects by nasal spray and aspiration

Objective Nasal washing (NW) is a popular method for collecting human nasal lavage fluid. However, for NW the subject must be trained, and the method is unsuitable for field studies on untrained subjects. To overcome this problem, we have developed an easy and painless method, a nasal spray and aspiration (NSA) method. Methods This method is different from NW in that the nasal cavity is misted over with saline, and the nasal lavage fluid is aspirated from the nostrils through a silicon tube. First, nasal lavage fluid was obtained twice by NSA with an interval of a week between lavages to evaluate intraindividual variability, and the IgA and protein levels in the nasal lavage fluid were measured by enzyme-linked immunosorbent assay and bicinchoninic acid assay, respectively. Next, the IgA value determined by NSA was compared with that by NW in another 12 normal subjects 2 days after NSA. Results In 10 normal subjects, mean volume of saline sprayed into the nose was 0.46 ± 0.15 ml (mean ± S.D.). Mean volume of aspirated nasal lavage fluid containing both sprayed saline and nasal secretion was 0.44 ± 0.37 ml. The mean IgA level/mg protein in the nasal lavage fluid determined by NSA was 112 ± 18 μg/mg protein at the first and 99 ± 20 at the second times of measurement, being highly reproducible. The mean value by NSA was 114 ± 19 μg/mg protein, being almost the same as that by NW of 99 ± 27. Conclusion These findings suggest that the IgA level/mg protein in nasal lavage fluid determined by NSA instead of NW might be useful for assessing the variability of nasal IgA secretion.

Changes of symptoms, tear film stability and eosinophilic cationic protein in nasal lavage fluid after re-exposure to a damp office building with a history of flooding

The aim was to study health effects in office workers (N = 18) in a medical case book archive with dampness caused by flooding. They were first investigated in a building without dampness (exposure free for 10 days). Then all returned to the damp building, and were re-investigated after 2 days. We measured tear film break up time (BUT), nasal patency, biomarkers in nasal lavage (NAL), and dynamic spirometry. Both buildings had low CO(2) (380-600 ppm), low levels of respirable particles (8-10 microg/m(3)), and formaldehyde (5-7 microg/m(3)). The flooded building had slightly higher (149 ng/m(3) vs. 94 ng/m(3)) levels of microbial volatile organic compounds (MVOC). After 2 days of re-exposure, there was an increase of ocular (P < 0.001), nasal (P = 0.002), and throat symptoms (P < 0.001), dyspnea (P = 0.006), headache (P = 0.002), nausea (P = 0.04), and tiredness (P = 0.01). The median BUT decreased from 16 to 8 s (P = 0.003), and eosinophilic cationic protein (ECP) in NAL increased slightly (P = 0.04). A separate test of the weekday effect showed slight improvements, or no change of symptoms and signs from Monday to Wednesday. In conclusion, subjects previously exposed to building dampness had an increase of symptoms, reduced tear film stability, and signs of eosinophilic inflammation in the nasal mucosa after 2 days of re-exposure. The study is in agreement with previous cross-sectional studies, suggesting that building dampness may cause mucosal irritation, general symptoms such as headache and tiredness, impaired tear film stability, and eosinophilic inflammation in the airway mucosa. From a preventive point of view, health consequences of water leakage and flooding should not be neglected. The measurements of molds and microbial volatile organic compounds could not identify any obvious exposure contrast between the damp building and the dry control building. This illustrates the limitations of air measurements of microbial exposures in damp buildings.

A Prospective, Randomized, Double-blind, Placebo-Controlled Multi-centre Study on the Efficacy and Safety of Sublingual Immunotherapy (SLIT) in Children With Seasonal Allergic Rhinoconjunctivitis to Grass Pollen

Purpose of the Study. Subcutaneous immunotherapy (SCIT) for seasonal allergic rhinitis is a well-established, effective, and potentially curative therapy. This study evaluated an alternate route for immunotherapy: the oral mucosa and gastrointestinal tract. Study Population. Ninety-seven children, aged 3 to 14 years, with seasonal allergic rhinitis and proven sensitivity to grass pollen were studied. Methods. Sensitivity to grass pollen was confirmed by positive skin-prick test, grass pollen–specific immunoglobulin E, and conjunctival provocation test. Patients were enrolled in a prospective, double-blind trial comparing sublingual immunotherapy (SLIT) to placebo. The treatment duration was 32 months. The primary outcome measure was the change in a multiple-symptom/medication score (which measured eye, nasal, and lung symptoms and rescue-medication use) after treatment. Data collected included patient-reported symptom scores and medication use, total and antigen-specific immunoglobulin E, skin-prick test, conjunctival provocation test, nasal provocation test, spirometry, exhaled nitric-oxide concentration, atopic dermatitis score, and eosinophilic cationic protein in nasal lavage fluid. Results. The multiple-symptom/medication score was significantly reduced by SLIT to 77.3% of the placebo group (P = .049). This overall score was affected mainly by a large reduction in rescue-medication usage among those in the treatment group (67% of placebo; P = .0025). There was no significant difference in any individual-symptom score. Conclusion. SLIT had a positive effect on rescue-medication usage but no significant effect on symptoms alone. Reviewer Comments. SLIT represents an alternative therapy with multiple potential advantages to SCIT, including the elimination of injections and improved safety profile. Several studies in adults and children have found improvement in symptom scores as well as reductions in medication use to the point where this is now being used in clinical practice in place of SCIT in many European countries. Additional studies, including investigation of optimal dosing and the potential to use multiple allergens, are needed to further define the future role of SLIT in the United States.

Psoriasin, one of several new proteins identified in nasal lavage fluid from allergic and non-allergic individuals using 2-dimensional gel electrophoresis and mass spectrometry

BackgroundExtravasation and luminal entry of plasma occurs continuously in the nose. This process is markedly facilitated in patients with symptomatic allergic rhinitis, resulting in an increased secretion of proteins. Identification of these proteins is an important step in the understanding of the pathological mechanisms in allergic diseases. DNA microarrays have recently made it possible to compare mRNA profiles of lavage fluids from healthy and diseased patients, whereas information on the protein level is still lacking.MethodsNasal lavage fluid was collected from 11 patients with symptomatic allergic rhinitis and 11 healthy volunteers. 2-dimensional gel electrophoresis was used to separate proteins in the lavage fluids. Protein spots were picked from the gels and identified using mass spectrometry and database search. Selected proteins were confirmed with western blot.Results61 spots were identified, of which 21 were separate proteins. 6 of these proteins (psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648) had not previously been described in nasal lavage fluids. The levels of psoriasin were markedly down-regulated in allergic individuals. Prolactin-inducible protein was also found to be down-regulated, whereas different fragments of albumin together with Ig gamma 2 chain c region, transthyretin and splice isoform 1 of Wnt-2B were up-regulated among the allergic patients.ConclusionThe identification of proteins in nasal lavage fluid with 2-dimensional gelelectrophoresis in combination with mass spectrometry is a novel tool to profile protein expression in allergic rhinitis and it might prove useful in the hunt for new therapeutic targets or diagnostic markers for allergic diseases. Psoriasin is a potent chemotactic factor and its down-regulation during inflammation might be of importance for the outcome of the disease.

Gene expression for NGF and NGF-receptors in the nasal mucosa of allergic rhinitic and control subjects

Rationale We were interested in the role of nerve growth factor (NGF) in augmented nasal reactivity to sensory irritants in seasonal allergic rhinitics (SAR). Previous investigators reported that SAR subjects had elevated NGF protein in nasal lavage fluid, but had lower mRNA levels in nasal scrapings, than non-rhinitic (NR) subjects. We compared the relative expression of genes coding for NGF, high-affinity receptor (TrkA/NTRK1) and low affinity receptor (p75/TNFRS16) in the nasal mucosa of SAR and NR subjects. Methods Total RNA was extracted from nasal scrapings from 15 non-asthmatic subjects, including 6 SAR and 9 NR controls. Sampling occurred outside of aeroallergen season. cDNA was synthesized from total RNA and used as templates for quantitative real-time PCR with TaqMan probes for NGF, TrkA/NTRK1, p75/TNFRS16 and GAPDH. Relative expression of NGF and NGF receptor genes was normalized to the expression of GAPDH and expressed in delta[CT] units. Results mRNA for NGF was of low abundance and detected in only 3/6 SARs and 6/9 NRs. Compared to NGF, there was a 5-fold higher relative expression of TrkA/NTRK1 and almost 300-fold higher expression of p75/TNFRS16. NR subjects had more NGF mRNA ( p<0.10) and significantly more mRNA for p75/TNFRS16 than SAR subjects ( p<0.05). mRNA for TrkA/NTRK1 did not differ significantly between sub-groups (p=0.57). Conclusions We observed decreased transcriptional message for both NGF and low affinity NGF receptor in superficial nasal scrapings of SAR vs NR subjects. This could reflect downregulation of NGF and p75/TNFRS16 in the superficial epithelium secondary to enhanced NGF production in the glandular compartment.

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 μg 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, α2-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.

In vivo conjugation of nasal lavage proteins by hexahydrophthalic anhydride

Hexahydrophthalic anhydride (HHPA), an industrially important chemical, is a highly allergenic compound. The aim of this work was to identify proteins in nasal lavage fluid (NLF) that form adducts with HHPA. Such bindings may induce production of specific immunoglobulin E (IgE) or affect physiological mechanisms of the proteins. NLF was obtained from HHPA-exposed volunteers, workers and exposed guinea pigs. HHPA-binding proteins were visualized with immunoblotting using a polyclonal antiserum against HHPA. The proteins were excised from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, digested with trypsin and identified by tandem mass spectrometry (MS/MS) and database searches. The antiserum was found to be specific for HHPA-bound proteins. In vivo formed HHPA-binding proteins in humans were identified as antileukoproteinase, immunoglobulin G (IgG), immunoglobulin A (IgA), serum albumin and lactoferrin. In addition, several proteins binding to HHPA were found in NLFs from guinea pigs but these could not be identified from database searches. Hypotheses for development of airways diseases by adduction of this allergenic compound to the NLF proteins in humans were established.

PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid.

PLUNC (palate, lung and nasal epithelial clone) is a newly discovered gene that is expressed in the upper respiratory tract and is suggested to be of importance in host defence against bacteria. We have identified two forms of the PLUNC protein in human nasal lavage fluid (NLF) using two-dimensional gel electrophoresis (2-DE) and MS. The apparent molecular masses and isoelectric points of these forms are 24.8 kDa/pI 5.4 and 25.1 kDa/pI 5.5. Notably, the 24.8 kDa/pI 5.4 form of PLUNC is an abundant protein in the 2-DE protein patterns of NLF from healthy subjects. Decreased levels of PLUNC were found in NLF from smokers and workers exposed to reactive epoxy chemicals, indicating that long-term exposure to airway irritants impairs the production of PLUNC in the upper respiratory tract. We have also investigated the presence of lipopolysaccharide (LPS)-binding proteins in NLF. Five proteins were found to adsorb to a LPS-coated surface; two of these proteins correspond to the two PLUNC forms, as judged by 2-DE pattern matching. For comparison, human saliva was found to contain a set of LPS-binding proteins with similar 2-DE spot positions (the same pIs but somewhat lower apparent molecular masses of approximately 20 kDa). These results indicate that PLUNC may be a new marker of airway inflammation and may play a part in the innate immune response, and that human saliva contains yet other members of the family of LPS-binding proteins.

Involvement of Eosinophils in the Early-Phase Allergic Reaction in a Guinea Pig Rhinitis Model

Background: Eosinophils are found in the nasal lavage fluid (NLF) and nasal biopsies of patients with allergic rhinitis after a nasal antigen challenge, and associated not only with a late-phase allergic reaction (LPR) but also an early phase allergic reaction (EPR). Numerous studies have been carried out to clarify the participation of eosinophils in LPR or airway hyperresponsiveness. However, there has been no published report describing in detail the role of eosinophils during EPR. To better understand the involvement of eosinophils in EPR, we studied the effects of repeated antigen challenges on nasal airway responsiveness and eosinophilic inflammation in EPR using a guinea pig rhinitis model. Methods: Nasal airway responsiveness was measured as the nasal airway resistance (NAR) after nasal antigen provocation. Eosinophilic inflammation during EPR was assessed by nasal lavage and histopathological examination using two groups of animals: those in group 1 were subjected to a sensitization pretreatment only, and those in group 2 were subjected to a pretreatment of sensitization followed by repeated nasal challenges. Results: Repeated antigen challenges induced nasal hyperresponsiveness as indicated by a decrease in the antigen provocation dose and a significant increase in NAR. Furthermore, significant increases in eosinophil counts, eosinophil peroxidase (EPO) activity and protein content in NLF during EPR were observed following antigen provocation in group 2. There were significant correlations between the levels of these parameters, and albumin was the most prevalent of the proteins in NLF. Histopathological examination showed that the degree of eosinophil infiltration into the lamina propria of the nasal mucosa of the animals in group 2 was significantly and apparently higher than in group 1. Particularly, epithelial disruption and mucosal edema were significantly elevated after antigen provocation in group 2. Conclusions: These results suggest that chronic eosinophil accumulation is induced by repeated antigen challenges in the nasal tissue, and that once antigen provocation occurs, eosinophils in the tissue are activated and responsible for the amplification of EPR such as vascular permeability and mucosal edema.

Newly identified proteins in human nasal and bronchoalveolar lavage fluids: potential biomedical and clinical applications.

Protein patterns of nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) were analyzed with two-dimensional gel electrophoresis (2-DE) and a number of previously unidentified proteins (lipocalin-1, cystatin S, transthyretin, immunoglobulin binding factor and an 11 kDa fragment of albumin) were identified by N-terminal amino acid sequencing. Lipocalin-1 was shown to be a dominant protein in NLF from healthy subjects but was almost undetectable in NLF from a patient with asthma. It further appeared that lipocalin-1 in NLF consists of eight forms with pIs between 5.2 and 5.5: three with the expected Mr of 17500, two with increased Mr (18000), and three truncated variants with Mr of 17000. Two forms of cystatin S were identified both in NLF and BALF: one with pI 5.1 and Mr 13000, and the other with pI4.9 and Mr 13500. The distribution of the two forms was clearly different in NLF and BALF from healthy subjects with the 4.9/13500 form constituting only about 13% in NLF but 69% in BALF. In NLF from subjects with upper airway irritation a twofold increased proportion of the 4.9/13500 form was detected. Amino acid sequence data and the spot position indicate that the 4.9/13500 form might be a phosphorylated variant of cystatin S. Lower levels of both forms of cystatin S were found in BALF from smokers than nonsmokers. The levels of transthyretin in NLF were decreased in subjects exposed to irritating chemicals. Finally, higher levels of IgBF were found in BALF from smokers than nonsmokers. Taken together, these results illustrate the potential biomedical and clinical applications of identifying proteins in 2-DE patterns of human BALF and NLF. The possibility to describe and monitor airway disorders at the molecular level is inferred.

The cellular basis for allergic rhinitis

In both seasonal and perennial rhinitis there is epithelial mast cell accumulation and tissue infiltration by eosinophils. Activation of these cells can be observed by electron microscopy and by elevated levels of tryptase and eosinophil cationic protein in nasal lavage fluid. Furthermore, seasonal increases in the antigen presenting cell (Langerhans' cell) are also evident. Investigations into the mechanisms involved in cell accumulation and activation reveals upregulation of leucocyte endothelial adhesion molecules and an increase in interleukin-4 (IL-4) in naturally occurring rhinitis, while mRNA for IL-4, IL-5 and granulocyte macrophage colony stimulating factor activity and lavage tumour necrosis factor-alpha (TNF alpha) levels are increased following local allergen challenge. These cytokines may be derived from a variety of sources, including mast cells, eosinophils and T-lymphocytes, and contribute to the underlying inflammatory process in rhinitis.

Basophils and eosinophils in allergic rhinitis

and HLA-DR positivity in the dermis. Arthritis Rheum 1988;31:672-7. 60. Seibold JR, Giorno RC, Claman HN. Dermal mast cell degranulation in systemic sclerosis. Arthritis Rheum 1990; 33:1702-9. 61. Chanez P, Lacoste J-Y, Guillot B, et al. Mast cells' contribution to the fibrosing alveolitis of the scleroderma lung. Am Rev Resp Dis 1993;147:1497-502. 62. de Paulis A, Valentini G, Spadaro G, Lupoli S, Tirri G, Marone G. Human basophil releasability. VIII. Increased basophil releasability in patients with scleroderma. Arthritis Rheum 1991;34:1289-96. 63. Kaufman LD, Gruber BL, Marchese M J, Seibold JR. Anti-IgE autoantibodies in systemic sclerosis (scleroderma). Ann Rheum Dis 1989;48;201-5. 64. Valent P, Bettelheim P. Cell surface structures on human basophils and mast cells: biochemical and functional characterization. Adv Immunol 1992;52:333-423. 65. Columbo M, Horowitz EM, Botana LM, et al. The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cell and peripheral blood basophils. J Immunol 1992;149:599-608. 66. de Paulis A, Ciccarelli A, Cirillo R, de Crescenzo G, Columbo M, Marone G. Modulation of human lung mast cell function by the c-kit receptor ligand. Int Arch Allergy Immunol 1992;99:326-9. 67. MacGlashan DW Jr, Hubbard WC. IL-3 alters free arachidonic acid generation in C5a-stimulated human basophils. J Immunol 1993;151:6358-69. 68. Cirillo R, Triggiani M, Siri L, et al. Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin. J Immunol 1990;144: 3891-7. 69. de Paulis A, Ciccarelli A, Cirillo R, de Crescenzo G, Columbo M, Marone G. Cytokine-induced modulation of human Fc,RI + inflammatory cells. In: Romagnani S, Mosmann TR, Abbas AK, eds. New advances on cytokines. New York: Raven Press, 1992:367-86. 70. Merget RD, Maurer AB, Koch U, et al. Histamine release from basophils after in vivo application of recombinant human interleukin-3 in man. Int Arch Allergy Appl Immunol 1990;92:366-74. 71. White MV, Igarashi Y, Emmery BE, Lotze MT, Kaliner MA. Effects of in vivo administration of interleukin-2 (IL-2) and IL-4, alone and in combination, on ex vivo human basophil histamine release. Blood 1992;76:1491-5.

Nasal challenge shows pathogenetic relevance of specific IgE serum antibodies for nasal symptoms caused by hexahydrophthalic anhydride

Hexahydrophthalic anhydride (HHPA) is a component of some epoxy resin systems. A high fraction of HHPA-exposed workers display nasal symptoms, and some of them have specific serum antibodies. To test the pathogenetic relevance of the antibodies nasal challenge tests were performed with a conjugate of HHPA and human serum albumin (HSA) at three increasing concentrations. Eleven subjects, who were IgE-sensitized against HHPA (positive in RAST and in skin-prick test against the HHPA-HSA conjugate), and who reported work-related nasal symptoms, had a significant increase of nasal symptoms and a decrease of nasal inspiratory peak flow after the challenges. The symptoms were associated with specific serum IgE, but with IgG. Further, significant increases were found in eosinophil and neutrophil counts, and in levels of tryptase, and albumin, whereas no clear rise was recorded for eosinophil cationic protein in nasal lavage fluid. Nine subjects, who were not sensitized, but who complained of work-related nasal symptoms, and 11 subjects, who were not sensitized and had no symptoms, displayed no significant change in any of these parameters. It is concluded that the symptoms in some of the workers were caused by an IgE-mediated mast cell degranulation, followed by an inflammatory reaction, engaging eosinophil and neutrophil cells.

Topical glucocorticoids inhibit activation by allergen in the upper respiratory tract

We have studied the effect of a topically administered glucocorticoid, fluticasone propionate (FP), on infiltration and activation of eosinophils in the nasal mueosa after provocation with allergen. Forty-four patients with seasonal allergic rhinitis entered a double-blind, crossover study in which they underwent treatment with either FP (200 μg once daily) or identical placebo for 2 weeks. Patients then underwent nasal-allergen provocation followed by nasal lavage and biopsy at one of several time points between 0 and 8 hours. Patients subsequently received the alternate treatment for 2 weeks before repeat allergen provocation, nasal lavage, and biopsy, as before. Biopsy specimens of nasal mueosa obtained during the immediate allergic response demonstrated an influx of eosinophils (stained by monoclonal antibody EG1) of similar magnitude during both FP and placebo treatment. Significantly, fewer esoinophils in these biopsy specimens were activated (stained by monoclonal antibody EG2) after treatment with FP compared with that after placebo treatment (median values, 8.8 and 36.6 cells per square millimeter, respectively; p < 0.02). The concentration of eosinophil cationic protein in nasal lavage fluid was significantly elevated above baseline from 2 to 8 hours after allergen, and this increase was abolished by treatment with FP. These results suggest that topical glucocorticoids inhibit allergen-induced activation of eosinophils in allergic rhinitis.