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1.
Lung ; 188(3): 209-16, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20082198

ABSTRACT

Our primary objective was to investigate the possible contribution of controller medications to asthmatic airway remodeling, by (1) comparing the apoptotic and necrotic effects of several corticosteroids and montelukast on cultured airway human bronchial surface epithelial (16HBE) and submucosal (Calu3) cells; (2) measuring epithelial shedding potential and desmosome length in response to a cytokine challenge, with or without co-administered corticosteroids; and (3) studying corticosteroids and montelukast effects on inter-cellular adhesion molecule (ICAM) expression in both 16HBE and human umbilical vein endothelial cells (HUVEC). For this purpose, apoptosis, necrosis, and ICAM expression were quantified by flow cytometry, with 16HBE cells sensitive to both the apoptotic and necrotic effects of dexamethasone and montelukast; Calu3 cells sensitive only to budesonide. Transmission electron microscopy revealed decreased desmosome length in the presence of cytokines (TNF-alpha and INF-gamma), with corticosteroids counteracting this reduction. Dexamethasone, beclomethasone, and montelukast decreased versus increased ICAM-1 expression in airway epithelial cells and HUVEC, respectively. For conclusions, bronchial surface epithelial and submucosal cells exhibit a different sensitivity profile toward dexamethasone, budesonide, and montelukast, with corticosteroids preventing cytokineinduced desmosomal damage in 16HBE cells. The studied drugs led to increased ICAM-1 expression in endothelium, potentially facilitating inflammatory cell migration into lung tissue.


Subject(s)
Acetates/pharmacology , Adrenal Cortex Hormones/pharmacology , Anti-Asthmatic Agents/pharmacology , Asthma/pathology , Endothelium, Vascular/drug effects , Leukotriene Antagonists/pharmacology , Quinolines/pharmacology , Respiratory Mucosa/drug effects , Acetates/therapeutic use , Adrenal Cortex Hormones/therapeutic use , Anti-Asthmatic Agents/therapeutic use , Apoptosis/drug effects , Asthma/drug therapy , Cell Line , Cyclopropanes , Cytokines/pharmacology , Desmosomes/drug effects , Endothelium, Vascular/pathology , Humans , Intercellular Adhesion Molecule-1/analysis , Leukotriene Antagonists/therapeutic use , Necrosis , Quinolines/therapeutic use , Respiratory Mucosa/pathology , Sulfides , Umbilical Veins/drug effects , Umbilical Veins/pathology
2.
Exp Mol Pathol ; 83(1): 47-53, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17383636

ABSTRACT

The aim of the study was to assess whether NCL-SG3, the only immortalized sweat gland cell line available, can be used as an in vitro model to study chloride ion transport in cultured sweat gland cells. Cl(-) efflux was measured using the MQAE dye fluorescence technique after stimulating the cells with different agonists. A significant stimulation of chloride efflux was achieved with the calcium ionophore A23187 resulting in an efflux rate of 0.9 mM/s. Both ATP and UTP activated chloride efflux in these cells, with the ATP response being larger. IBMX and forskolin stimulation did not induce a rate of chloride efflux above the basal level. Immunocytochemistry showed no detectable CFTR in NCL-SG3 cells. This finding was confirmed with flow cytometry analysis. Niflumic acid (20 and 100 microM NFA) and 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (100 ìM) decreased the rate of ATP-stimulated chloride efflux significantly (0.40 and 0.31 mM/s with NFA, 0.37 mM/s with H2DIDS). Gadolinium (20 ìM) had no effect on the chloride transport rate. In conclusion, the NCL-SG3 cells retain some of the aspects of human sweat gland epithelium, such as the ability to form cell-cell contacts. The CFTR protein is neither functional nor expressed in cultured NCL-SG3 sweat gland cells. Ca(2+)-activated chloride conductance is confirmed and the putative Ca(2+)-activated chloride channel (CaCC) is further characterized in term of its pharmacological sensitivity. The NCL-SG3 sweat gland cell line can be used to investigate the characteristics of the CaCC and to identify the channel.


Subject(s)
Chloride Channels/metabolism , Chlorides/metabolism , Sweat Glands/metabolism , Biomarkers , Calcium/metabolism , Cell Line , Epithelial Cells/metabolism , Humans , Immunohistochemistry , Ion Channel Gating , Ion Transport , Microscopy, Electron, Transmission , Sweat Glands/ultrastructure
3.
Microsc Res Tech ; 69(4): 271-6, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16586482

ABSTRACT

The ionic composition of the airway surface liquid (ASL) in healthy individuals and in patients with cystic fibrosis (CF) has been debated. Ion transport properties of the upper airway epithelium are similar to those of the lower airways and it is easier to collect nasal ASL from the nose. ASL was collected with ion exchange beads, and the elemental composition of nasal fluid was determined by X-ray microanalysis in healthy subjects, CF patients, CF heterozygotes, patients with rhinitis, and with primary ciliary dyskinesia (PCD). In healthy subjects, the ionic concentrations were approximately isotonic. In CF patients, CF heterozygotes, rhinitis, and PCD patients, [Na] and [Cl] were significantly higher compared when compared with those in controls. [K] was significantly higher in CF and PCD patients compared with that in controls. Severely affected CF patients had higher ionic concentrations in their nasal ASL than in patients with mild or moderate symptoms. Female CF patients had higher levels of Na, Cl, and K than male patients. As higher salt concentrations in the ASL are also found in other patients with airway diseases involving chronic inflammation, it appears likely that inflammation-induced epithelial damage is important in determining the ionic composition of the ASL.


Subject(s)
Body Fluids/chemistry , Cystic Fibrosis/metabolism , Kartagener Syndrome/metabolism , Nasal Lavage Fluid/chemistry , Nasal Mucosa/chemistry , Rhinitis/metabolism , Adolescent , Adult , Child , Chlorine/analysis , Electron Probe Microanalysis , Female , Heterozygote , Humans , Male , Middle Aged , Potassium/analysis , Salts/analysis , Sex Factors , Sodium/analysis
4.
Respir Med ; 100(11): 2018-28, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16580832

ABSTRACT

Nasal polyposis is a poorly understood chronic inflammatory disease often associated with asthma. As nasal polyps and asthma both are associated with massive eosinophil infiltration, they may share a common pathophysiological mechanism. Many genetic and autoimmune diseases may result from altered expression or function of cell adhesion molecules such as desmosomes. A transmission electron microscopical study was carried out on tissue from 15 patients suffering from nasal polyps, to investigate if there are changes in desmosomes in nasal polyps from asthmatic and/or allergic patients versus non-asthmatic versus non-allergic patients. In allergic patients the damage to columnar cells was more extensive than in non-allergic patients. Massive infiltration of eosinophils was observed in epithelium and connective tissue in all groups. No significant difference in thickness of the basal lamina was found between any of the groups. All patients had dilated capillaries in the connective tissue. The intercellular space between the epithelial cells was smallest in the asthmatic non-allergic group. The relative length of columnar cell or basal cell desmosomes was reduced in patients with asthma or allergy, compared to non-allergic, non-asthmatic patients. Hence, there appears to be a weakness in the desmosomes in asthmatics and allergics. Epithelial shedding may play an important role in the pathophysiological process of a multifactorial disease such as asthma.


Subject(s)
Asthma/pathology , Nasal Polyps/ultrastructure , Adult , Aged , Asthma/complications , Basement Membrane/ultrastructure , Connective Tissue/ultrastructure , Desmosomes/ultrastructure , Eosinophils/ultrastructure , Epithelial Cells/ultrastructure , Female , Humans , Male , Microscopy, Electron , Middle Aged , Nasal Polyps/complications
5.
Respir Med ; 99(4): 429-43, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15763449

ABSTRACT

Epithelial damage is commonly found in airways of asthma patients. The aim of this study was to investigate epithelial damage in allergic and non-allergic asthma at the ultrastructural level. Bronchial biopsies obtained from patients with allergic asthma (n=11), non-allergic asthma (n=7), and healthy controls (n=5) were studied by transmission electron microscopy. Epithelial damage was found to be extensive in both asthma groups. Both in basal and in columnar cells, relative desmosome length was reduced by 30-40%. In columnar cells, half-desmosomes (i.e., desmosomes of which only one side was present) were frequently noticed. Eosinophils showing piece-meal degranulation were commonly observed in allergic asthma. Degranulating mast cells were more often observed in allergic asthma. Goblet cell hyperplasia was only found in allergic asthma. Lymphocytes were increased in both groups. In both groups, the lamina densa of the basal lamina was thicker than the control by about 40-50%. In allergic asthma the lamina densa was irregular with focal thickening. While there was always a tendency for changes (epithelial damage, desmosomes, degranulating mast cells, basal lamina) to be more extensive in allergic asthma compared to non-allergic asthma, there was no significant difference between the two groups in this respect. Reduced desmosomal contact may be an important factor in the epithelial shedding observed in patients with asthma.


Subject(s)
Asthma/pathology , Bronchi/ultrastructure , Adult , Basement Membrane/ultrastructure , Biopsy/methods , Bronchoscopy/methods , Desmosomes/ultrastructure , Eosinophils/ultrastructure , Female , Goblet Cells/ultrastructure , Humans , Lymphocytes/ultrastructure , Male , Mast Cells/ultrastructure , Microscopy, Electron, Transmission , Respiratory Mucosa/ultrastructure
6.
Cell Biol Int ; 28(5): 397-402, 2004.
Article in English | MEDLINE | ID: mdl-15193283

ABSTRACT

One of the mechanisms that has been put forward for the development of the androgen-resistant status is neuroendocrine differentiation. Neuroendocrine cells secrete neuropeptides that may represent one of the possible molecular bases by which hormone-dependent prostate cancer cells could escape treatment. LNCaP prostate cancer cells were treated with either etoposide or neuropeptides. Morphological changes related to apoptosis and cell viability were assessed. Changes in intracellular ion content were quantitatively analyzed by electron probe X-ray microanalysis. Etoposide treatment consistently induces a decrease in K and an increase in Na, which are inhibited by bombesin or calcitonin. The Na/K ratio increased markedly after exposure to etoposide, and both bombesin and calcitonin blocked this increase. Etoposide also caused changes in the intracellular P and S concentrations that to a large extent could be blocked by neuropeptides. These results support the hypothesis that neuropeptides confer anti-apoptotic capabilities onto non-neuroendocrine cells in close proximity to neuroendocrine cells.


Subject(s)
Apoptosis/drug effects , Etoposide/antagonists & inhibitors , Neuropeptides/pharmacology , Potassium/metabolism , Prostatic Neoplasms/metabolism , Protective Agents/pharmacology , Sodium/metabolism , Cell Division/drug effects , Cell Line, Tumor , Cell Shape/drug effects , Cell Survival/drug effects , Electron Probe Microanalysis/methods , Etoposide/pharmacology , Humans , Kinetics , Male , Microscopy, Electron, Scanning/methods , Prostatic Neoplasms/drug therapy
7.
Histol Histopathol ; 19(3): 951-61, 2004 07.
Article in English | MEDLINE | ID: mdl-15168357

ABSTRACT

It has been suggested that neuroendocrine (NE) cells provide paracrine stimuli for the propagation of local carcinoma cells and that NE differentiation is associated with the progression of prostate cancer toward an androgen-independent state. Apoptosis comprises a critical intracellular defense mechanism against tumorigenic growth and is associated with a number of changes in the elemental content of the cell. The neuropeptides bombesin and calcitonin, which inhibit etoposide-induced apoptosis, also inhibit the etoposide-induced elemental changes in prostate carcinoma cells. This important fact strengthens the link between apoptosis and changes in the intracellular elemental content. This protective effect on etoposide-induced apoptosis appears to be quite similar in androgen-dependent and androgen-independent cell lines. This confirms that neuropeptides confer antiapoptotic capabilities on non-neuroendocrine cells in close proximity to neuroendocrine cells. It can therefore be speculated that certain neuroendocrine peptides can increase the survival and further growth of neighboring cells and may thereby contribute to the aggressive clinical course of prostate tumors containing neuroendocrine elements. In addition, this correlation provides an objective basis for the study of neuropeptide target points and may be helpful for alternative therapeutic protocols using neuropeptide inhibitors in the treatment of patients with advanced prostatic carcinoma. The culture techniques described were, thus, designed in order to achieve two important goals. First, the development of an in vitro model that allows an approach to neuroendocrine differentiation in prostate cancer and its role in apoptosis blockage. Second, the method has been designed in order to permit rapid cryofixation of intact cell monolayers for subsequent x-ray microanalysis.


Subject(s)
Apoptosis/drug effects , Ions/metabolism , Neuropeptides/pharmacology , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Cell Line, Tumor , Electron Probe Microanalysis , Humans , Male , Microscopy, Electron, Scanning , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/ultrastructure
8.
Eur Respir J ; 20(6): 1444-8, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12503702

ABSTRACT

Asthmatics are known to react to inhaled hyperosmolar solution. Therefore, the effect of hyperosmolar salt solutions on tight junctions of the airway epithelium was investigated by electron microscopy. Rat trachea was perfused with different concentrations of sodium chloride (NaCl) and then fixed from the luminal side with glutaraldehyde to which the electron dense tracer lanthanum chloride had been added. Lanthanum penetrated 3+/-1% of the tight junctions in trachea perfused with 295 mOsm Krebs-Ringer's buffer (KRB). Adding NaCl to the KRB (KRB-NaCl) increased osmolarity of the solution. After perfusion with 589 or 876 mOsm KRB-NaCl, lanthanum was observed in the lateral intercellular spaces in 50+/-11 and 57+/-6%, respectively. The effect of hyperosmolarity was reversible and only 6+/-1% of the tight junctions were penetrated after perfusion with 295 mOsm KRB solution following 589 mOsm KRB-NaCl perfusion. Adding mannitol to the KRB to an osmolarity of 589 mOsm only caused 5+/-1% of the tight junctions to open, even though osmotic effects were observed. Opening the tight junctions with hyperosmolar salt solutions may play a role in exercise-induced asthma. It may also open the prospect for increased penetration of inhaled drugs into the interstitium and the circulation.


Subject(s)
Saline Solution, Hypertonic/pharmacology , Tight Junctions/drug effects , Trachea/ultrastructure , Animals , Lanthanum/pharmacology , Microscopy, Electron , Permeability , Rats , Rats, Sprague-Dawley
9.
Clin Sci (Lond) ; 103(4): 417-24, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12241542

ABSTRACT

Cystic fibrosis is a heterogenic disease, in which the phenotype can also vary for patients with the same genotype. In the present study the function of the cystic fibrosis transmembrane conductance regulator (CFTR) in nasal epithelial cells from 19 adult patients with cystic fibrosis was investigated. All patients had severe mutations, whereby no or little functional CFTR is expected in the plasma membrane. Of the patients, 15 were homozygous for deltaF508-CFTR (i.e. CTFR lacking residue Phe-508). The others were deltaF508-heterozygous with 3659delC, 394delTT or 2183AA-->G. Nasal epithelial cells, obtained by nasal brushings, were loaded with the fluorescent probe N -(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide to measure Cl(-) efflux. In most of the cystic fibrosis patients, forskolin plus isobutylmethylxanthine was unable to elicit any response. Unexpectedly, cells from three cystic fibrosis patients (two deltaF508/deltaF508 patients and one deltaF508/3659delC patient) responded to stimulation in a wild-type manner. It was investigated whether this residual chloride transport function was associated with a milder phenotype. Clinical parameters studied were lung function, number of antibiotic courses, Shwachman score, Bhalla score, age at chronic colonization with Pseudomonas aeruginosa and the pattern of essential fatty acids in serum phospholipids. Unknown factors may affect the presence of functional CFTR in patients with severe CFTR mutations. However, we could not find a correlation between the response to cAMP and any of the phenotype parameters. It appears that functional cAMP transport in the nasal epithelium is no guarantee of a mild phenotype and, conversely, that a patient lacking cAMP-dependent chloride transport can develop a mild phenotype.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Nasal Mucosa/metabolism , Adolescent , Adult , Cells, Cultured , Chlorides/metabolism , Cyclic AMP/pharmacology , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epithelial Cells/metabolism , Female , Genotype , Humans , Male , Mutation , Phenotype , Severity of Illness Index
10.
Histol Histopathol ; 17(1): 165-77, 2002 01.
Article in English | MEDLINE | ID: mdl-11813866

ABSTRACT

The effects of irradiation on intestinal epithelial cells were analyzed in vivo and in vitro. The in vivo study was carried out on the rat small intestine and for the in vitro study the intestinal crypt cell-line IEC-6 was used. Rat intestine and IEC-6 cells were irradiated with X-ray doses ranging between 1-16 Gy. Energy-dispersive X-ray microanalysis was used for detection of the elemental changes in the cells. Cell morphology was investigated in the scanning electron microscope, DNA-synthesis by autoradiography of 3H-thymidine incorporating nuclei and proliferation by cell counting. Our results indicate that in vivo, in the crypt cells, the increasing doses of irradiation led to increased sodium and lowered potassium and phosphorus concentrations. Corresponding ion shifts were found in the irradiated IEC-6 cells. Cells continued to proliferate up to the dose of 8 Gy, although the proliferation rate became lower with increasing dose of irradiation. The increasing dose of irradiation significantly reduced DNA-synthesis (16 Gy decreased DNA-synthesis by 50%) which resulted in a complete inhibition of cell proliferation. Analysis of goblet cells also showed characteristic radiation-dependent elemental changes. Scanning electron microscopical investigation of cells in culture revealed that most of the control cells were flat and had rather smooth cell membranes. Irradiation led to the appearance of numerous different membrane manifestations (microvilli of varying length and distribution, and blebs). Frequency of differences in the topology of the cells was related to the dose of irradiation. Our study clearly demonstrates that even low doses of irradiation cause changes in the ionic composition of the cells and inhibit DNA-synthesis and cell proliferation. The effects observed in the crypt cells in vivo were the same as in the intestinal cell line in vitro, which indicates that IEC-6 cells can be used for investigation of side effects of radiation to the abdomen.


Subject(s)
Epithelial Cells/radiation effects , Intestine, Small/cytology , Intestine, Small/radiation effects , Animals , Cell Division/radiation effects , Cell Line , Cell Nucleus/ultrastructure , Cells, Cultured , Cytoplasm/ultrastructure , DNA/biosynthesis , DNA/radiation effects , Electron Probe Microanalysis , Epithelial Cells/metabolism , Intestine, Small/metabolism , Male , Microscopy, Electron, Scanning , Rats , Rats, Sprague-Dawley , Tissue Fixation
11.
J Submicrosc Cytol Pathol ; 34(4): 381-8, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12575837

ABSTRACT

The effects of nicotine on intestinal and tracheal mucosa and epithelial cells were studied in vivo and in vitro. Rats received 108 microM nicotine in their drinking water for 10 days. Intestine and trachea were removed and prepared for X-ray microanalysis, transmission electron microscopy and immunocytochemistry. X-ray microanalysis of freeze-dried cryosections of intestine and trachea showed an increase in Na and Cl, and a decrease in K in the lamina propria of the intestine and the epithelial cells and the submucosal compartment of the trachea. Analysis of frozen-hydrated trachea, in order to determine ionic changes in the airway-surface liquid (ASL), indicated that the Na and Cl content in the ASL decreased after nicotine treatment. Immunocytochemistry showed upregulation of ICAM-1 in the submucosal connective tissue of the trachea and the lamina propria of the intestine. Transmission electron microscopy showed a somewhat increased number of eosinophils in the lamina propria of nicotine-treated rats, increased edema in the submucosal connective tissue, and a somewhat increased number of damaged basal cells in the trachea of nicotine-treated rats, compared to the controls. These data indicate that nicotine may evoke an inflammatory reaction, in particular in the trachea, that could cause cell damage and with that changes in the ionic relations of the epithelial cells. The in vitro experiments showed that nicotine could directly affect ion transport by inhibiting cAMP-stimulated (but not ATP-stimulated) chloride efflux from cultured respiratory epithelial cells. This indicates that apart from indirect effects via inflammation, nicotine can directly affect the ionic homeostasis of the cells and the composition of the airway-surface liquid.


Subject(s)
Intestinal Mucosa/drug effects , Nicotine/pharmacology , Respiratory Mucosa/drug effects , Trachea/drug effects , Administration, Oral , Animals , Biological Transport, Active , Cell Line , Chlorides/analysis , Chlorides/metabolism , Electron Probe Microanalysis , Immunohistochemistry , Intestinal Mucosa/metabolism , Intestinal Mucosa/ultrastructure , Ion Transport , Male , Microscopy, Electron , Nicotine/administration & dosage , Potassium/analysis , Potassium/metabolism , Rats , Rats, Wistar , Respiratory Mucosa/metabolism , Respiratory Mucosa/ultrastructure , Sodium/analysis , Sodium/metabolism , Trachea/pathology , Water Supply
12.
Eur Cell Mater ; 3: 1-8, 2002 Mar 18.
Article in English | MEDLINE | ID: mdl-14562254

ABSTRACT

X-ray microanalysis is commonly applied in biomaterials research to study changes in biomaterial composition, calcifications, or to identify particulates in tissue that has been in contact with biomaterials. Studies where the effect of biomaterials on the naturally occurring elements in the cells are studied are rare. Exposure to or contact with biomaterials may give rise to several cellular reactions with characteristic changes in elemental patterns. Experimental systems in which cultured cells are exposed to biomaterials, and the resulting changes in elemental content measured by X-ray microanalysis are presented. Proliferation results in an increase in Mg, P and K. Injury and necrosis result in increased Na, Cl and Ca, and decreased K and Mg. The Na/K ratio is a sensitive indicator of cell damage. Apoptosis results in increased Na/K and an increase P/S ratios. Mechanical effects of biomaterials may cause changes in cell-shape or cell volume that may result in volume-regulating ion fluxes, e.g., of chloride, accompanied by efflux of potassium. The multi-element detection capability of X-ray microanalysis makes it particularly suitable to detect these characteristic patterns of changes. The technique can also be used to define sub populations of cells that differ with respect to their reactions to external stimuli.

13.
Respir Med ; 95(11): 904-10, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11716205

ABSTRACT

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.


Subject(s)
Bronchial Hyperreactivity/pathology , Sjogren's Syndrome/pathology , Adult , Biopsy/methods , Bronchial Hyperreactivity/complications , Bronchial Provocation Tests , Bronchoconstrictor Agents , Case-Control Studies , Eosinophils/immunology , Female , Humans , Interleukin-4/immunology , Interleukin-5/immunology , Interleukin-8/immunology , Laminin/analysis , Male , Mast Cells/immunology , Methacholine Chloride , Middle Aged , Neutrophils/immunology , Sjogren's Syndrome/complications , Statistics, Nonparametric , T-Lymphocytes/immunology , Tenascin/analysis
14.
J Microsc ; 203(Pt 3): 277-84, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11555145

ABSTRACT

The genetic disease cystic fibrosis (CF) is due to defective epithelial chloride transport. Different treatments have been proposed that could restore chloride transport in CF patients. A new method is proposed for measuring the chloride secretion in easily accessible epithelial cells. Fresh nasal epithelial cells were obtained by nasal brushing and made to attach to titanium grids for electron microscopy. Chloride efflux through the cystic fibrosis transmembrane regulator channel was stimulated by 20 microM forskolin and 100 microM isobutyl-methylxanthine (IBMX), in standard Ringer's solution (SR). Chloride efflux through the calcium-regulated channel was stimulated by 200 microM adenosine triphosphate (ATP) in SR. The cells were rinsed after the exposure, in order to remove the experimental medium, frozen and freeze-dried. The elemental composition of the cells was determined by X-ray microanalysis. Rinsing with distilled water or ammonium acetate appeared to cause damage to the cells, whereas rinsing with isotonic mannitol preserved the ionic composition. Stimulation of cells from healthy controls with forskolin and IBMX in a chloride-containing medium caused a significant (28 +/- 6%) decrease in chloride concentration, which is indicative of net chloride efflux. In similar conditions, stimulation with ATP induced a 29 +/- 5% decrease in the chloride concentration. Stimulation of cells from CF patients with forskolin and IBMX in a chloride-containing medium caused no significant change in the intracellular chloride concentration, whereas ATP stimulation induced a response similar to that obtained in cells from healthy controls. It is concluded that X-ray microanalysis of nasal epithelial cells may be used to determine chloride secretion in CF patients in an easily accessible cell type.


Subject(s)
Chlorides/metabolism , Cystic Fibrosis/metabolism , Electron Probe Microanalysis/methods , Epithelial Cells/metabolism , Nasal Mucosa/metabolism , 1-Methyl-3-isobutylxanthine/pharmacology , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Colforsin/pharmacology , Cystic Fibrosis/pathology , Epithelial Cells/drug effects , Epithelial Cells/pathology , Humans , Ion Transport/drug effects , Nasal Mucosa/drug effects , Nasal Mucosa/pathology
15.
Microsc Res Tech ; 54(2): 114-22, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11455618

ABSTRACT

The synchronous contractions of the uterus in labour depend on electrical coupling of myometrial smooth muscle cells by gap junctions. In the human myometrium, gap junctions are scarce in the non-pregnant uterus, but become abundant at term in preparation for labour. We have previously demonstrated that in the human myometrium at term, three different gap-junctional proteins are expressed, connexins 43, 45, and 40. These connexins are known to have distinctive functional capacities in in vitro expression systems but whether, in the human myometrium in vivo, they are co-assembled into the same gap junction or form different types of gap junction has previously been unclear. By applying triple immunogold labelling to sections of Lowicryl-embedded tissue for electron microscopy, together with complementary immunoconfocal microscopy, we demonstrate here that connexins 43, 45, and 40 are commonly present as mixtures within the same gap-junctional plaque. While all gap junctions contain connexin43, the relative signal for each connexin type varies between individual junctions. The presence within single gap-junctional plaques of three different connexins, each with the potential for conferring distinctive channel properties, suggests an inherent versatility for modulation of smooth muscle cell intercellular communication properties during human parturition.


Subject(s)
Connexins/metabolism , Gap Junctions/metabolism , Myometrium/ultrastructure , Animals , Female , Guinea Pigs , Humans , Immunohistochemistry , Microscopy, Confocal , Myometrium/metabolism , Pregnancy , Rabbits
16.
Cell Biol Int ; 25(6): 563-6, 2001.
Article in English | MEDLINE | ID: mdl-11407863

ABSTRACT

Proliferation and death of airway epithelial cells may be of importance in the pathogenesis of asthma. T-helper (Th)-2 response interleukins (IL)-4, -5, and -13 have the ability to induce proliferation of airway epithelial cells.


Subject(s)
Interleukins/pharmacology , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Th2 Cells/immunology , Asthma/immunology , Bronchi/cytology , Cell Division/drug effects , Cell Division/immunology , Cell Survival/drug effects , Cell Survival/immunology , Cytotoxicity Tests, Immunologic , Humans , Interleukin-13/pharmacology , Interleukin-4/pharmacology , Interleukin-5/pharmacology , Respiratory Mucosa/drug effects
17.
Cell Biol Int ; 25(6): 499-508, 2001.
Article in English | MEDLINE | ID: mdl-11407855

ABSTRACT

Apoptosis comprises a critical intracellular defense mechanism against tumourigenic growth. We have been interested in the relationship between morphological changes and intracellular concentration of several cations after etoposide-induced apoptosis in androgen-independent prostate cancer cells. SEM and X-ray microanalysis were performed on freeze-dried PC3 cells after etoposide treatment, and correlated with the morphological features observed after examination by light and fluorescence microscopy. Cell viability assays were also performed. A significant decrease in intracellular Cl(-) and K(+)and a progressive increase in Mg(2+) and Na(+) were observed, with parallel changes in cellular volume as cells passed through three morphological stages of apoptosis. The use of EPXRMA made it possible to evaluate alterations in element composition in prostate cancer cell apoptosis and may be a helpful tool for further studies on apoptosis in prostate cancer.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/physiology , Electron Probe Microanalysis , Etoposide/pharmacology , Prostatic Neoplasms , Apoptosis/drug effects , Cell Division/drug effects , Cell Size/drug effects , Cell Survival/drug effects , DNA, Neoplasm/analysis , Flow Cytometry , Humans , Male , Microscopy, Electron, Scanning , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/ultrastructure
18.
Free Radic Res ; 34(5): 499-511, 2001 May.
Article in English | MEDLINE | ID: mdl-11378533

ABSTRACT

Neutrophils and eosinophils are involved in the pathogenesis of many respiratory diseases. The enzymes myeloperoxidase and eosinophil peroxidase catalyze the reaction of H2 O2 with Cl to produce the reactive oxygen species HOCl. Normal human bronchial epithelial (NHBE) cells were exposed to 0.18-0.90 mM HOCl for 48 h, and studied with immunohistochemical, metabolic and morphological studies. The ability of the cells to attach to each other and/or to the matrix was altered. Immunohistochemical studies showed a decreased amount of desmosomes and focal adhesion sites, although the morphology of the cells was not affected. The ability of the mitochondria to oxidize glucose was reduced. HOCl-exposed cells had an increased production of NO, probably by an increased activity of cNOS, due to increased intracellular Ca2+. The antioxidant N-acetylcysteine inhibited both the NO production and the effects of HOCl on glucose oxidation. The cNOS-inhibitor N-propyl-L-arginine inhibited HOCl-induced NO production. X-ray microanalysis showed an increase in the intracellular Na+ /K+ ratio, which indicates cell damage. In conclusion, exposure to HOCl results in cell detachment and metabolic alterations in normal human bronchial epithelial cells. Oxygen radicals could in part mediate the effects. Oxygen radicals could hence contribute to the observed epithelial damage in respiratory diseases.


Subject(s)
Bronchi/cytology , Bronchi/drug effects , Hypochlorous Acid/pharmacology , Bronchi/metabolism , Calcium/metabolism , Cell Adhesion/drug effects , Cell Survival/drug effects , Cells, Cultured , Cytosol/drug effects , Cytosol/metabolism , Desmosomes/drug effects , Electron Probe Microanalysis , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Free Radicals/metabolism , Glucose/metabolism , Humans , NF-kappa B/drug effects , NF-kappa B/metabolism , Nitric Oxide Synthase/drug effects , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitrites/metabolism , Oxidation-Reduction , Vinculin/metabolism
19.
Int J Biochem Cell Biol ; 33(4): 315-24, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11312102

ABSTRACT

Vascular endothelial growth factor (VEGF-A) exerts its effects through receptor tyrosine kinases VEGF receptor-1 (VEGFR-1) and VEGFR-2, which are expressed on most endothelial cell types in vitro and in vivo. We have examined VEGF-A-induced signal transduction in porcine aortic endothelial (PAE) cells individually expressing VEGFR-1 or VEGFR-2, and cells co-expressing both receptor types. We show that VEGF-A-stimulated PAE cells co-expressing VEGFR-1 and -2 contain receptor heterodimers. VEGF-A-stimulation of all three cell lines (expressing VEGFR-1, -2 and -1/2) resulted in signal transduction with different efficiencies. Thus, tyrosine phosphorylation of phospholipase Cgamma, and accumulation of inositol polyphosphates were efficiently transduced in the VEGFR-1/2 cells whereas cells expressing VEGFR-1 responded poorly in these assays. In contrast, VEGF-A-induced activation of phosphoinositide 3-kinase and induction of Ca2+ fluxes were transduced well by VEGFR-1 and VEGFR-2 homo- and heterodimers. The pattern of Ca2+ fluxes was unique for each type of VEGF receptor dimer. Our data show that signal transduction induced by VEGF-A is transduced in distinct manners by homo- and heterodimers of VEGF receptors.


Subject(s)
Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/metabolism , Signal Transduction , Amino Acid Sequence , Animals , Cell Line , Dimerization , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Enzyme Activation , Inositol Phosphates/metabolism , Isoenzymes/metabolism , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/metabolism , Phospholipase C gamma , Proto-Oncogene Proteins/chemistry , Receptor Protein-Tyrosine Kinases/chemistry , Receptors, Growth Factor/chemistry , Receptors, Vascular Endothelial Growth Factor , Substrate Specificity , Swine , Type C Phospholipases/metabolism , Vascular Endothelial Growth Factor Receptor-1
20.
J Allergy Clin Immunol ; 107(2): 249-57, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11174190

ABSTRACT

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.


Subject(s)
Epithelial Cells/pathology , Rhinitis, Allergic, Perennial/pathology , Rhinitis/pathology , Adult , Antibodies, Monoclonal , Cell Count , Cell Degranulation/immunology , Eosinophils/cytology , Eosinophils/immunology , Female , Humans , Inflammation/pathology , Male , Mast Cells/cytology , Mast Cells/immunology , Middle Aged , Nasal Mucosa/cytology , Neutrophils/cytology , Neutrophils/immunology , Time Factors
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