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Biomedical subjects

L Sevéus

Publications and source records attributed to L Sevéus.

17 recordsLinked to original sources
PubMed2002

Identification of intracellular markers in induced sputum and bronchoalveolar lavage samples in patients with respiratory disorders and healthy persons.

Induced sputum and bronchoalveolar lavage (BAL) are widely used for retrieving cells and soluble materials for studies of airway inflammation. Centrifuged cell samples are suitable for immunochemical identification of cellular products. The aim was to determine the optimal fixation procedure to detect intracellular antigens in situ. In immunocytochemistry, an appropriate choice of fixation method is a prerequisite for identification of cells and, consequently, for reliability results. We compared eight fixation and permeabilization methods to detect intracellular antigens in cytocentrifuged cell samples. Four granular proteins specific to eosinophils (eosinophil cationic protein, ECP; eosinophil peroxidase, EPO) and neutrophils (human neutrophil lipocalin, HNL; myeloperoxidase, MPO) were the antigens studied. We found that the organic solvents often used in immunocytochemistry are unsuitable fixatives for detection of these intracellular low-molecular-weight proteins. Treatment with crosslinking fixatives alone resulted in incomplete penetration of antibodies into the cell interiors. Best results were obtained using a commercial reagent Ortho PermeaFix (OPF) for flow cytometry. With this, fixation and permeabilization take place simultaneously OPF-treated cells retained their structural characteristics, and the antibodies studied penetrated both cellular and granule membranes. With OPF treatment, ECP EPO, HNL, and MPO were fixed on their places in granules, and their antigenicity was retained. Correct identification of intracellular proteins is important in characterization of the respiratory inflammatory response in clinical work and research.

Acute-Phase Proteins↗
PubMed2002

Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis.

BACKGROUND AND AIM: Accumulation and infiltration by neutrophil granulocytes is a prominent feature in the local inflammatory process in ulcerative colitis (UC). The present study was performed to evaluate human neutrophil lipocalin (HNL) as a specific neutrophil marker in the inflamed lesions of the colon and rectum in patients with colitis and proctitis. METHODS: The activity of intestinal neutrophils with respect to release of granule proteins was studied in 18 patients with UC (10 with colitis and eight with isolated proctitis) and in 18 healthy controls using perfusion fluid and biopsies from the sigmoid colon and rectum. The released amounts of the neutrophil granule proteins HNL and myeloperoxidase (MPO) were determined by radioimmunoassays, and the location of HNL and MPO in biopsies from colonic mucosa was examined by immunohistochemistry. RESULTS: Mucosal release of HNL and MPO was increased 10-55-fold in patients with colitis and proctitis compared with controls. Their bowel biopsies demonstrated that only neutrophils were stained with anti-HNL. We also found correlations between HNL and levels of granulocyte/macrophage-colony stimulating factor (GM-CSF) and interleukin 8 (IL-8) in perfusion fluids from the sigmoidal segments of patients with proctitis, between HNL and GM-CSF in rectal segments in patients with proctitis, and in sigmoidal segments in patients with colitis. CONCLUSION: We conclude that the increased release of HNL and MPO in colorectal perfusion fluids indicates neutrophil involvement in the local inflammatory process, and suggest that HNL may serve as a specific marker of intestinal neutrophil activation in UC. GM-CSF, and to some extent IL-8, may play a role in neutrophil accumulation and priming in this disease.

Acute-Phase Proteins↗
PubMed2002

Cell specific markers for eosinophils and neutrophils in sputum and bronchoalveolar lavage fluid of patients with respiratory conditions and healthy subjects.

BACKGROUND: Highly specific protein markers for eosinophils and neutrophils could be a valuable diagnostic aid in various respiratory disorders. The cell specificity of monoclonal antibodies against eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), human neutrophil lipocalin (HNL), and myeloperoxidase (MPO) was investigated using immunocytochemical techniques. METHODS: Induced sputum and bronchoalveolar lavage fluid samples from 14 patients with respiratory conditions and four healthy individuals were studied. Antigens were detected at their intracellular sites in cells with well preserved structures using optimal techniques for fixation, permeabilisation, and immunolabelling. RESULTS: Anti-EPO antibodies reacted only with eosinophils, and anti-HNL antibodies only with neutrophils. Anti-ECP antibodies reacted with both eosinophils and neutrophils and anti-MPO antibodies with neutrophils and monocytes. Cells not stained by monoclonal anti-EPO and anti-HNL antibodies included lymphocytes, monocytes, macrophages, squamous epithelial cells, and ciliated epithelial cells. CONCLUSIONS: EPO, a unique component of eosinophils, and HNL, a unique component of neutrophils, are useful markers for the identification of eosinophils and neutrophils, respectively, in sputum and bronchoalveolar lavage fluid.

Acute-Phase Proteins↗
PubMed2001

Inflammation and structural changes in the airways of patients with primary Sjögren's syndrome.

The present study aimed to compare the cellular pattern and structural changes in the airways of patients with primary Sjögren's syndrome (pSS) with healthy controls. Bronchial biopsy specimens were obtained from seven subjects with pSS and seven healthy controls. All the patients with pSS had increased bronchial responsiveness to methacholine. In the biopsies inflammatory cells, cytokine-producing cells, tenascin and laminin were visual zed by immunostaining. Patients with pSS had a higher number of neutrophils and mast cells than healthy controls, while the number of eosinophils was similar in the two groups. The number of IL-8-positive cells was higher in pSS butthe numbers of IL-4-and IL-5-positive cells were not significantly different between pSS and healthy controls. The numbers of T cells in patients with pSS were higher than in healthy controls, while the numbers of CD25-positive cells were similar to the healthy controls. The degree of epithelial integrity in patients with pSS was significantly lower than in the control group and the tenascin and laminin layers were significantly thicker in the pSS group. There was a correlation between the number of mast cells and the thickness of the tenascin and laminin layers in pSS. In conclusion, we found that the cellular pattern in the bronchial mucosa of patients with pSS displayed large numbers of neutrophils, mast cells and T-lymphocytes. These changes in inflammatory cell numbers seemed to relate to the observed increased epithelial damage and structural changes of the subepithelium. The structural findings, but not the pattern of inflammatory cells, are shared with atopic asthma and may relate to the increased bronchial hyper-responsiveness seen in both diseases.

Adult↗
PubMed2001

Inflammatory cell and epithelial characteristics of perennial allergic and nonallergic rhinitis with a symptom history of 1 to 3 years' duration.

BACKGROUND: Perennial rhinitis is an inflammatory condition of the mucosal lining of the nose that may be caused by allergic and nonallergic mechanisms. OBJECTIVE: We sought to characterize the cellular pattern and structural changes in the nasal mucous membrane of patients with perennial rhinitis and compare them with those of control subjects. METHODS: Biopsy specimens were obtained from 27 patients with perennial allergic rhinitis (PAR), from 12 patients with perennial nonallergic rhinitis (PNAR) with eosinophils present in the nasal smear, and from 6 control subjects without rhinitis. In 10 of 27 patients with PAR who were also allergic to pollen, biopsy specimens were taken within the respective season (PARseason). In the other 17 patients, the biopsy was taken outside the pollen season (PARoutside season). Inflammatory cells were identified by using mAbs to their unique granular proteins. RESULTS: The characteristic feature of perennial rhinitis was the accumulation of activated (degranulated) mast cells and eosinophils in the nasal mucosa. The tissue eosinophil/neutrophil ratio was higher, and the loss of epithelial integrity was greater in all patient groups compared with the control subjects. The extent of epithelial damage was significantly larger in patients in the PARseason group compared with that in the PARoutside season and PNAR groups, which did not significantly differ from each other in this respect. The number of eosinophils and mast cells was higher in the PNAR group compared with the PAR groups. In all patient groups, the number of eosinophils correlated with the loss of epithelial integrity. The number of mast cells did not correlate with the extent of epithelial damage nor did the number of neutrophils, except in patients in the PARseason group. CONCLUSION: The accumulation of eosinophils and mast cells, as well as loss of epithelial integrity, was characteristic for perennial rhinitis. Loss of epithelial integrity in the nasal mucosa may be a consequence of the activity of accumulated eosinophils.

Adult↗
PubMed2000

Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. BHR Group.

The aim of the present study was to compare the cellular pattern and structural changes in the airway walls of atopic and nonatopic patients with asthma. Bronchial biopsy specimens were obtained from 13 atopic subjects with asthma, nine nonatopic patients with asthma, and seven healthy control subjects and investigated using immunohistochemical methods. The number of eosinophils increased in both asthma groups, but significantly more in the atopic group. The number of mast cells increased similarly in the two asthma groups, whereas the number of neutrophils increased only in the nonatopic asthma group. The number of T-lymphocytes (CD3-, CD4-, CD8-, CD-25-positive cells) was higher in patients with atopic asthma compared with nonatopic asthma. Interleukin-4 (IL-4) and IL-5-positive cells were more frequently found in the atopic asthma group, whereas cells staining for IL-8 were more frequent in the nonatopic group. The degree of epithelial damage was significantly higher in the atopic asthma group compared with the control subjects and the nonatopic asthmatics. The tenascin and laminin layer was significantly thicker in the atopic group compared with the group of nonatopic asthmatics. In the atopic group, there was a significant negative correlation between epithelial integrity (defined as the relative length of intact epithelium) and the eosinophil count and also between the number of CD25-positive cells and epithelial integrity. The number of mast cells correlated positively with the thickness of tenascin- and laminin-positive layers. In conclusion, we provide evidence of different patterns of involvement of inflammatory cells in atopic and nonatopic patients with asthma. There were also structural differences in the bronchial mucous membrane between atopic asthma and nonatopic asthma. This suggests that there are differences in the extent of the immunopathologic response of these clinically distinct forms of asthma.

Adolescent↗
PubMed2000

Piecemeal degranulation of peripheral blood eosinophils: a study of allergic subjects during and out of the pollen season.

The variability of serum and plasma levels of eosinophil granule proteins in different clinical conditions, interpreted as the result of different patterns of cytokine priming, suggests a selective mobilization of granule proteins. Inasmuch as piecemeal degranulation (PM) is the mechanism proposed for the differential release of eosinophil granule proteins, we decided to investigate whether blood eosinophils from allergic subjects show characteristics of PM during natural allergen challenge. Eosinophils from three birch-sensitive subjects were studied before and during the pollen season. Electron microscopy analysis showed that during the season, eosinophils presented morphologic features of PM. By immunogold labeling, eosinophil cationic protein (ECP) was detected not only in normal specific granules but also in the cytoplasm, in the vicinity of partially lucent specific granules. These results were confirmed by subcellular fractionation, where the amount of ECP associated with compartments containing small vesicles increased 2-fold during the pollen season. A study of the distribution of ECP, eosinophil peroxidase, and hexosaminidase in eosinophils of different densities showed that the profile of each of these proteins differed depending on cell density. All of these proteins decreased in the specific granule of hypodense cells and increased in other cell compartments. We conclude that allergen exposure causes PM of the peripheral blood eosinophils of allergic subjects, and that the density of these cells reflects the degree of degranulation. Our results provide novel information for the understanding of the selective mobilization of granule proteins into the circulation.

Blood Proteins↗
PubMed1999

Mast cells and tryptase in the middle ear of children with otitis media with effusion.

OBJECTIVES: This is the first report of the presence of tryptase, a reflection of mast cell activity, in chronic middle ear effusion of patients in whom atopic status was characterized. DESIGN AND METHODS: Mediator activity of mast cells and eosinophils was measured prospectively from effusion of 33 random patients and five controls with chronic otitis media with effusion (OME). Atopy was determined by enzyme-linked immunosorbent assay testing. Middle ear biopsies from a second group of eight OME patients and four controls were fixed in plastic and stained immunohistochemically for mast cells. RESULTS: Sixty-one percent of patients had extensive activation of mast cells in their middle ears. Among those with elevated tryptase in their effusion, 95.6% were atopic and 94.7% also had elevated effusion eosinophilic cationic protein. Tryptase was elevated only in the effusion of atopic patients as compared with controls (P < 0.01). Mast cells were present in six of eight OME ears and absent in all four normals. CONCLUSION: Mast cells and its mediator tryptase, both indicators of a Th2-driven immune response, are present in the majority of ears which have chronic effusion, suggesting that the inflammation within the middle ear of most OME patients is allergic in nature.

Adolescent↗
PubMed1999

Eosinophils and neutrophils in biopsies from the middle ear of atopic children with otitis media with effusion.

OBJECTIVE AND DESIGN: The majority of patients with otitis media with effusion (OME) and atopy have been shown to have elevated levels of eosinophil cationic protein (ECP) in their middle ear fluid. The mechanism underlying these elevated levels of ECP is not clear. The purpose of this study was to investigate the feasibility of a quantitative determination of eosinophils and neutrophils in the middle ear lining by specific immunocytochemical markers, in order to study the extent of the involvement of these cells in patients with OME. METHODS: Bilateral middle ear biopsies from five children with persistent OME and atopy confirmed by in vitro testing were evaluated for the presence of eosinophils and neutrophils with monoclonal antibodies against specific granule proteins. Five subjects who had no signs of effusion or infection but were undergoing routine tympanoplasty for dry perforations served as controls. The biopsies were embedded in a plastic resin to improve the structural preservation of the target cells and to increase the resolution in the light microscope. Dual markers were used to determine which marker was better for eosinophils and neutrophils, respectively. The following markers were used: eosinophil cationic protein (EG2), and eosinophil peroxidase (EPO) for eosinophils and myeloperoxidase (MPO) and human neutrophil lipocalin (HNL) for neutrophils. RESULTS: Antibodies against EPO gave a more localized and intense staining than antibodies against EG2. Antibodies against HNL appear more specific to neutrophils than antibodies against MPO that also recognize monocytes. The number of cells was determined both in the tissue and in the mucus covering the epithelium. Eosinophils and neutrophils were present in the subepithelial connective tissue and in the mucus blanket in the middle ear of patients with OME in significantly higher number than in the control group. In general, there were more inflammatory cells in the mucus than in the tissue itself, but the number of inflammatory cells in the mucus showed a significant positive correlation with the number of inflammatory cells in the tissue. There was a significant positive correlation between the number of neutrophils and the number of eosinophils in the tissue as well as in the mucus, irrespective of which marker was used. CONCLUSION: The results of this study show the feasibility of using specific antibodies to identify eosinophils and neutrophils in the middle ear. The initial data suggest that atopic children with OME have higher numbers of such cells as compared to non-OME controls.

Adolescent↗
PubMed1999

Evidence of mast cell activity in the middle ears of children with otitis media with effusion.

OBJECTIVES: This is the first study to report the presence of tryptase, a reflection of mast cell activity, in chronic middle ear effusion of patients whose atopic status was characterized. DESIGN AND METHODS: Mediator activity of mast cells and eosinophils was measured prospectively from effusion of 33 randomly selected patients and 5 control subjects with chronic otitis media with effusion (OME). Atopy was determined by enzyme-linked immunosorbent assay. Middle ear biopsies from a second group of 8 OME patients and 4 controls were fixed in plastic and stained immunohistochemically for mast cells. RESULTS: Sixty-one percent of patients had extensive activation of mast cells in their middle ears. Among those with elevated tryptase in their effusion, 95.6% were atopic and 94.7% also had elevated levels of effusion eosinophilic cationic protein (ECP). Tryptase levels were elevated only in the effusion of atopic patients, as compared with 5 controls (P < .01). Mast cells were present in 6 of 8 OME ears and absent in all 4 normal ears. CONCLUSION: Mast cells and its mediator tryptase, both indicators of a Th2-driven immune response, are present in a majority of ears that have chronic effusion. These findings support the hypothesis that middle ear mucosa is capable of an allergic response and that the inflammation within the middle ear of most OME patients is allergic in nature.

Adolescent↗
PubMed1997

Human neutrophil lipocalin (HNL) is a specific granule constituent of the neutrophil granulocyte. Studies in bronchial and lung parenchymal tissue and peripheral blood cells.

The neutrophilic granulocyte is a cytotoxic and potentially tissue-injuring cell participating in the destructive processes and symptoms seen in a variety of inflammatory diseases. Sensitive immunoassays have been introduced to measure the levels of specific secretory proteins of various inflammatory cells in blood and other body fluids. The aim has been to develop highly specific markers for each cell type. The results obtained by immunoassay have indicated that human neutrophil lipocalin (HNL) is a protein unique to the neutrophil. The present study investigated the specificity of HNL as a neutrophil marker in peripheral blood and lung tissue by using flow cytometry and immunocytochemistry. Flow cytometry and immunocytochemistry on peripheral blood showed that monoclonal antibodies to HNL only react with neutrophils and not with other types of leukocytes. Immunocytochemistry on plastic-embedded sections and on frozen sections of lung tissue showed that a cocktail of six monoclonal antibodies to HNL specifically reacts with neutrophils and not with epithelial cells or macrophages. By immunoelectron microscopical studies performed on healthy human neutrophils after low temperature embedding in Lowicryl K4M following aldehyde fixation and partial dehydration, it could be shown that HNL colocalized with lactoferrin (a known marker for secondary or specific granules) and that myeloperoxidase was localized in the primary or azurophil granules. The results confirm that HNL is a unique component of the secondary granules of the neutrophil granulocyte.

Acute-Phase Proteins↗
PubMed1994

Use of fluorescent europium chelates as labels in microscopy allows glutaraldehyde fixation and permanent mounting and leads to reduced autofluorescence and good long-term stability.

In the present study europium chelates were introduced as alternative fluorescent labels for microscopy and their effect on enhanced autofluorescence caused by the glutaraldehyde fixative was investigated. Glutaraldehyde fixation was used to stabilize the cells for a permanent mount after the immunocytochemical reaction. The europium signal in time-resolved fluorescence microscopy was shown to be free of autofluorescence when strong cross-linking fixation with glutaraldehyde was used and the signal-to-background ratio obtained was 2,400 or better. It was also shown that the europium signal was stable in daylight and at room temperature. Fluorescent europium chelate used in this experiment provides excellent contrast and long-term stability for the samples with glutaraldehyde fixation and permanent mounting.

Antibodies, Monoclonal↗
PubMed1978

On the occurence and composition of dense particles in mitochondria in ultrathin frozen dry sections.

Clusters of dense particles were observed irregularly in mitochondrial profiles of shock-frozen brown adipose tissue and liver when cryosectioned at a chamber temperature of -80 degrees C and not if sectioning was performed at -110 degrees C. The particles are rich in Ca and P (Ca/P =0.5); they are therefore not any form of tricalcium orthophosphate (e.g. hydroxyapatite). They apparently form by electrolyte diffusion in the section and therefore cannot be considered as representing the in vivo pool of mitochondrial calcium.

Adipose Tissue, Brown↗
PubMed1978

Preparation of biological material for X-ray microanalysis of diffusible elements. I. Rapid freezing of biological tissue in nitrogen slush and preparation of ultrathin frozen sections in the absence of trough liquid.

Any method used for the analysis of diffusible elements within a cell should involve conditions which eliminate the redistribution of those elements. Redistribution of diffusible substances is caused by (a) ice damage during freezing, (b) thawing during sectioning, and (c) thawing during handling of sections. To eliminate this sections were cut (a) in the 5-8 micrometer ice crystal free surface layer obtained by freezing in melting nitrogen, (b) at a specimen temperature of 133 K and a knife temperature of 173 K to 153 K, and the temperature of the chamber was kept low by evaporating nitrogen from an extra reservoir inside the chamber, by regulating the output of a foil heater. A grid carrier was designed to simplify the section collection and the sections were affixed on to the grid in a press assembly. The sections were hard and translucent, and showed faint interference colours.

Adipose Tissue, Brown↗
PubMed1978

Preparation of biological material for X-ray microanalysis of diffusible elements. II. Comparison of different methods of drying ultrathin cryosections cut without a trough liquid.

The appearance of ultrathin, dry-cut cryosections of brown adipose tissue and liver was found to be strongly dependent upon adequate freeze-drying. If freeze-drying was inadequate, diffusion of substances could be demonstrated and freezing damage was not apparent. Diffusion was manifested as an electron dense film over structural features such as triglyceride droplet profiles and the edges of the section; when this film was thick, X-ray signals for P, S, Cl, K and Ca could be detected from it, in different proportions to those found in the section. The frequency and intensity of diffusion were lowered by decreasing the temperature in the cryochamber from about 200 K +/- 5 K to 163 K +/- 5 K by forced evaporation of liquid N2 using an extra heater. The lowest incidence of diffusion was obtained in conjunction with this device, by leaving the sections in the cryochamber for at least 2 h during drying, either over a drying agent or under moderate vacuum. Such sections showed a narrow zone (A) at the edge that lacked obvious ice-crystal damage, a thicker zone (B) of moderate ice-crystal damage and the bulk of the interior (zone C) severely damaged by freezing. Qualitatively different, reproducible X-ray spectra could be obtained from ultrastructural features even in areas showing some signs of diffusion.

Adipose Tissue, Brown↗
PubMedDate not supplied

Cryoultramicrotomy as a preparative method for x-ray microanalysis in pathology.

The role of ion shifts in cell injury has become one of the most intriguing fields of application of X-ray microanalysis in pathology. In principle, cryo(ultra)-microtomy is the best preparative method for X-ray microanalysis of diffusible substances. In this review, the following points are discussed: the choice of freezing method and coolant, the use of high molecular weight polymer cryoprotectants and their possible physiological effects the choice of the cryosectioning temperature, and techniques for handling and transfer of sections. Experiences with the sectioning of cryoprotected tissue are presented. Finally, other preparative techniques for microanalysis are compared to cryoultramicrotomy.

Animals↗