Göran Karlsson

The cellular response of the human allergic mucosa to natural allergen exposure.

It has been suggested that the IgE-dependent late-phase reaction to allergen exposure, with the features of an inflammatory cellular infiltration and airway hyperreactivity, is a link between anaphylaxis and continuous allergic airway disease. Our main knowledge of the cellular response to allergen in sensitized individuals has been derived from allergen-challenge models. To explore the dynamics of the cellular response during the actual disease, patients with a strictly seasonal allergic rhinitis were studied during natural allergen exposure. Ten patients suffering from an isolated birch-pollen allergy were followed from a symptom-free state before, during, and to the height of the birch-pollen season. Repeated parallel cell samplings from the nasal mucosa were performed with cytologic imprints on plastic strips, nasal lavages with the recovery of the cells in the lavage fluid with cytocentrifugation on object slides for cytologic study, and scrapings from the nasal surface with a curette for histologic and ultrastructural evaluation. The histamine content was determined in lavage fluid and cell pellets. The tosyl-alpha-tosyl-L-arginine methyl esterase activity of the nasal lavage fluid was also determined as a biochemical marker of the allergic inflammatory reaction. The birch-pollen season was moderate in terms of pollen counts, and this resulted in mild to moderate nasal symptoms that ran parallel to the birch-pollen counts. The total number of cells recovered in the lavage fluid was 1.2 +/- 0.4 (SEM) x 10(6) before and 3.2 +/- 2.0 per 10(6) cells (not significant) during pollen exposure. Most cells were neutrophils and mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cells and eosinophils in the allergic mucosal response to allergen challenge: Changes in distribution and signs of activation in relation to symptoms☆

An allergen challenge was performed in 10 asymptomatic patients with strictly seasonal allergic rhinitis. For comparison; seven nonallergic subjects were challenged with allergen, and seven allergic patients were challenged with diluent. Cell samples, obtained with use of a brush technique to recover cells from within the epithelium and nasal lavage to collect cells from the epithelial surface, and symptom scores were taken before challenge and at 2-hour intervals during 12 hours. The cell suspensions were cytocentrifuged onto object slides for light microscopy. Histamine was determined in the cell pellets. In brush samples from the allergic patients challenged with allergen, eosinophils, expressed as a percentage of the total granulocytes, increased from 4.3% +/- 2.7% (mean +/- SEM) to 10.3% +/- 3.8% (p < 0.05) 4 hours after challenge. This level was maintained for up to 12 hours. A similar increase was noted in the lavage specimens 2, 6, and 8 hours after the challenge. In the brush samples the proportion of eosinophils containing two or more cytoplasmic vacuoles, taken as a sign of activation, increased from 20% to 72% (p < 0.05) 8 hours after provocation. In brush samples from the allergic patients challenged with allergen, the numbers of metachromatic cells increased to a maximum of eightfold at 10 hours. In the lavage specimens, no metachromatic cells were observed before provocation, but they progressively increased in number 2 to 12 hours after provocation. Cell pellet histamine content decreased temporarily 2 to 4 hours after challenge (p < 0.05) in brush samples from allergen-challenged allergic patients. The local metachromatic cell density before challenge, as reflected in the brush specimens, correlated with nasal congestion, sneezing, and the degree of eosinophilia.

A brush method to harvest cells from the nasal mucosa for microscopic and biochemical analysis

A method is described for the sampling of epithelial cells and other effector cells from the human airway mucosa for structural and biochemical analysis. The cell samples are obtained from the nasal mucosa using a small nylon brush which is rotated over the epithelium and soaked and shaken in a small volume of a balanced salt solution. Morphological evaluation using light microscopy and transmission electron microscopy revealed excellently preserved cytological detail. In asymptomatic individuals the cells harvested were as follows: 45 +/- 5.9% (mean +/- SEM) epithelial cells, 38 +/- 7.1% granulocytes, 16 +/- 2.3% large mononuclear cells (monocytes), and 1.3 +/- 2.3% eosinophils. Repeated measurements in the same individual revealed a coefficient of variation of the order of 40% for the proportions of cells harvested. In comparison with nasal airway lavage, a higher proportion of epithelial cells and monocytes were obtained with the brush method. The cells harvested could also be used for biochemical analysis. The histamine content of the cell pellets was found to be strongly correlated with the mast cell count (r = 0.93) and was estimated to about 10 pg/cell, which is higher than previously reported for mast cells obtained from human lung tissue dispersed by an enzymatic method. The present method appears to be appropriate for the study of cellular events in the nasal mucosal epithelium.

Does Allergic Rhinitis Predispose to Sinusitis

The relationship between allergic rhinitis and sinusitis is reviewed with regard to seasonal as well as perennial and fungal sinusitis. There seems to be some association between allergic processes in the nose and inflammation in the sinus mucosa but the available data are, to some extent, contradictory. Thus, the causative role of allergy in sinusitis is not clear. Prospective studies to further elucidate the influence of allergic inflammation in the pathogenesis of sinusitis are highly warranted.

Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.

Proton-translocating transhydrogenase: an update of unsolved and controversial issues

Proton-translocating transhydrogenases, reducing NADP+ by NADH through hydride transfer, are membrane proteins utilizing the electrochemical proton gradient for NADPH generation. The enzymes have important physiological roles in the maintenance of e.g. reduced glutathione, relevant for essentially all cell types. Following X-ray crystallography and structural resolution of the soluble substrate-binding domains, mechanistic aspects of the hydride transfer are beginning to be resolved. However, the structure of the intact enzyme is unknown. Key questions regarding the coupling mechanism, i.e., the mechanism of proton translocation, are addressed using the separately expressed substrate-binding domains. Important aspects are therefore which functions and properties of mainly the soluble NADP(H)-binding domain, but also the NAD(H)-binding domain, are relevant for proton translocation, how the soluble domains communicate with the membrane domain, and the mechanism of proton translocation through the membrane domain.

Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 silica column at 55 degrees C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149-->Asp149 or Asn152-->Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time.

Increased nasal mucosal swelling in subjects with asthma

Objective The objective of this study was to evaluate nasal mucosal swelling with acoustic rhinometry in subjects with asthma and in healthy controls.

Mast cells and mediators in the nasal mucosa after allergen challenge. Effects of four weeks' treatment with topical glucocorticoid

The study focuses on the relationship between the tissue density of mast cells, the tissue hislamine levels and the levels of markers of mast cell activation after an allergen challenge of the nasal mucosa of allergic patients. The effect of 4 weeks treatment with a topical glucocorticoid, fluticasone propionate, was studied in a double‐blind, placebo‐controlled study of 25 hay fever patients. Nasal biopsies were obtained before and after the treatment period for the evaluation of mast cell density and tissue histamine levels. Nasal challenges were performed at 2‐week intervals for 8 weeks using a standardized nasal lavage model. TAME‐esterase was analysed in the returned lavage fluid from all the challenges (weeks 0–8), while the levels of histamine and tryptase were analysed in lavage fluids from challenges performed before and after the treatment period (weeks 0 and 4). The symptoms of nasal allergy were assessed after each challenge. Treatment with fluticasone proprionate did not influence mast cell density, the tissue histamine concentration, the lavage histamine levels or the TAME‐esterase activity, while a reduction in nasal symptoms and tryptase in nasal lavagc fluid was revealed. Our present study again emphasizes the fact that the mast cell is an important trigger cell in the immediate nasal allergic response. The study also demonstrates the usefulness of the measurements of tryptase as an indicator of both mast cell activation and the efficacy of topical steroid treatment.

A mutation in POLE predisposing to a multi-tumour phenotype

Somatic mutations in the POLE gene encoding the catalytic subunit of DNA polymerase ɛ have been found in sporadic colorectal cancers (CRCs) and are most likely of importance in tumour development and/or progression. Recently, families with dominantly inherited colorectal adenomas and colorectal cancer were shown to have a causative heterozygous germline mutation in the proofreading exonuclease domain of POLE. The highly penetrant mutation was associated with predisposition to CRC only and no extra-colonic tumours were observed. We have identified a mutation in a large family in which the carriers not only developed CRC, they also demonstrate a highly penetrant predisposition to extra-intestinal tumours such as ovarian, endometrial and brain tumours. The mutation, NM_006231.2:c.1089C>A, p.Asn363Lys, also located in the proofreading exonuclease domain is directly involved in DNA binding. Theoretical prediction of the amino acid substitution suggests a profound effect of the substrate binding capability and a more severe impairment of the catalytic activity compared to the previously reported germline mutation. A possible genotype to phenotype correlation for deleterious mutations in POLE might exist that needs to be considered in the follow-up of mutation carriers.