Bengt Ståhlbom

Newly identified proteins in human nasal lavage fluid from non-smokers and smokers using two-dimensional gel electrophoresis and peptide mass fingerprinting.

Human nasal lavage fluids (NLFs) were analyzed with two‐dimensional gel electrophoresis (2‐DE) and proteins were identified with peptide mass fingerprinting using matrix‐assisted laser desoption/ionization time of flight mass spectrometry. In some cases, the identification was verified by analysis of post‐source decay fragmentation spectra. Many of the identified proteins were new forms or fragments of previously found proteins (e.g. albumin, lactoferrin, cystatin, calgranulin, von Ebners gland protein and palate lung nasal epithelium clone), while others were proteins that have previously been indicated by 2‐DE image matching or immunoblots (e.g. apolipoprotein AI, lysozyme C, and Clara cell secretory protein). Some new proteins, not shown before in 2‐DE patterns of NLF were also found, e.g. mammaglobin B, 2‐microglobulin and immunoglobulin J chain. Of the identified NLF proteins many appear to be involved in inflammatory and immune responses. A study was therefore conducted to investigate if the levels of these proteins were changed in smokers compared to nonsmokers. It was found that NLF from smokers contained decreased levels of Clara cell secretory protein, and increased proportions of a truncated variant of lipocortin‐1, three acidic forms of α1‐antitrypsin, and one phosphorylated form of cystatin S. Furthermore, NLF from smokers contained increased proportions of a new variant of palate lung nasal epithelium clone (PLUNC), a recently identified airway irritation marker. The results demonstrate that 2‐DE of NLF may be used to assess alterations of proteins or post‐translationally modified proteins in smokers. Clara cell secretory protein (CC 16, CC 10) and lipocortin‐1 are two anti‐inflammatory, phospholipase A2 inhibitory proteins, and α1‐antitrypsin and cystatin S are two proteinase inhibitors. Changed levels of these proteins may therefore be of importance to the airway inflammation caused by smoking. The results also support the notion that PLUNC is involved in inflammatory responses in the upper airways.

Identification of a new potential airway irritation marker, palate lung nasal epithelial clone protein, in human nasal lavage fluid with two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time of flight.

We have analyzed protein patterns of human nasal lavage fluid (NLF) with two‐dimensional gel electrophoresis (2‐DE) and identified several proteins (such as transthyretin, Clara Cell protein 16, lipocalin‐1, cystatin S, cystatin SN, immunoglobulin binding factor, statherin, calgranulin B, prolactin‐inducible protein, and zinc‐α2‐glycoprotein) by N‐terminal amino acid sequencing and matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry. To investigate whether airway irritation causes alterations in NLF 2‐DE patterns, we compared epoxy workers with airway irritation (n=8) and healthy controls (n=6) before and after 2 h exposure to the epoxy chemical, dimethylbenzylamine (DMBA, 100 νg/m3) in an exposure chamber. A 25 kDa protein with pI 5.5 was found to be altered in the NLF 2‐DE patterns; a trypsin digest of the 2‐DE spot analyzed by MALDI‐TOF and expressed sequence tags (ESTs) determined after post‐source decay (PSD) identified the protein as palate lung and nasal epithelial clone (PLUNC). In controls, the levels of NLF‐PLUNC were generally lower after 2 h exposure, whereas in epoxy workers, the levels were increased three‐ to twentyfold after exposure. The human gene sequence for PLUNC was just recently reported and so far no biofunctional data are available. Our results suggest that PLUNC is involved in the airway inflammatory response after exposure to irritants.

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 ap peared that lipocalin‐1 in NLF consists of eight forms with pIs between 5.2 and 5.5: three with the expected Mr of 17 500, two with increased Mr (18 000), and three truncated variants with Mr of 17 000. Two forms of cystatin S were identified both in NLF and BALF: one with pI 5.1 and Mr 13 000, and the other with pI 4.9 and Mr 13 500. The distribution of the two forms was clearly different in NLF and BALF from healthy subjects with the 4.9/13 500 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 4.9/13 500 form was detected. Amino acid sequence data and the spot position indicate that the 4.9/13 500 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.

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.

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.

A prospective study of the relationship between exposure and specific antibodies in workers exposed to organic acid anhydrides.

Background: The exposure–response relationships for the induction of specific IgE and IgG were evaluated in a prospective study of workers exposed to organic acid anhydrides (OAAs). Special attention was paid to the modifying effects of atopy and smoking.

Trimethylamine and trimethylamine oxide levels in normal women and women with bacterial vaginosis reflect a local metabolism in vaginal secretion as compared to urine

The smell of rotten fish is one of the characteristics of bacterial vaginosis (BV), and is due to trimethylamine (TMA). Trimethylamine can be found in human urine, although most of it occurs as the nonvolatile oxide (TMAO) form. The fraction TMA/TMAO can be expected to be the same in different body fluids if no local production of TMA occurs. In women with BV, TMAO in the vaginal fluid is expected to be chemically reduced by the local bacterial flora to the much more odorous TMA. We have therefore studied the local vaginal production of TMA in vaginal secretion compared to the general TMA‐TMAO metabolism that was measured in urine using gas chromatography. Both vaginal fluid and random urine samples were collected from women, with and without BV, attending a Swedish clinic for sexually transmitted diseases, and these samples were analyzed for TMA and TMAO. The results show that a local production of TMA occurs in the vagina that is not part of the general metabolism of TMA‐TMAO.

Evaluating Measuring Techniques for Occupational Exposure during Additive Manufacturing of Metals: A Pilot Study

Additive manufacturing that creates three-dimensional objects by adding layer uponlayer of material is a new technique that has proven to be an excellent tool for themanufacturing of complex struct ...