Christer Tagesson

Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease

Background: Crohns disease is associated with deranged intestinal permeability in vivo, suggesting dysfunction of tight junctions. The luminal contents are important for development of neoinflammation following resection. Regulation of tight junctions by luminal factors has not previously been studied in Crohns disease. Aims: The aim of the study was to investigate the effects of a luminal stimulus, known to affect tight junctions, on the distal ileum in patients with Crohns disease. Patients: Surgical specimens from the distal ileum of patients with Crohns disease (n=12) were studied, and ileal specimens from colon cancer patients (n=13) served as controls. Methods: Mucosal permeability to 51Cr-EDTA and electrical resistance were studied in Ussing chambers during luminal exposure to sodium caprate (a constituent of milk fat, affecting tight junctions) or to buffer only. The mechanisms involved were studied by mucosal ATP levels, and by electron and confocal microscopy. Results: Baseline permeability was the same in non-inflamed ileum of Crohns disease and controls. Sodium caprate induced a rapid increase in paracellular permeability—that is, increased permeation of 51Cr-EDTA and decreased electrical resistance—which was more pronounced in non-inflamed ileum of Crohns disease, and electron microscopy showed dilatations within the tight junctions. Moreover, sodium caprate induced disassembly of perijunctional filamentous actin was more pronounced in Crohns disease mucosa. Mucosal permeability changes were accompanied by mitochondrial swelling and a fall in epithelial ATP content, suggesting uncoupling of oxidative phosphorylation. Conclusions: The tight junctions in the non-inflamed distal ileum of Crohns disease were more reactive to luminal stimuli, possibly mediated via disturbed cytoskeletal contractility. This could contribute to the development of mucosal neoinflammation in Crohns disease.

Determination of urinary 8-hydroxydeoxyguanosine by automated coupled-column high performance liquid chromatography : A powerful technique for assaying in vivo oxidative DNA damage in cancer patients

An automated analytical method has been developed for determination of the oxidative DNA adduct, 8-hydroxydeoxyguanosine (8OHdG) in human urine, based on coupled-column high performance liquid chromatography with electrochemical detection. Urine is concentrated on Bondelut CH by means of an automated sample processor, and the enriched sample injected on to a polymeric reversed phase column coupled in line with an electrochemical detector and a C18 reversed phase column. By use of the electrochemical detector, a suitable retention time interval is set for collection of the fraction containing 8OHdG from the chromatography on the first column; this fraction is collected in a 2 mL loop and injected onto the C18 column. The system is operated by an automatic valve station controlled by an integrator. The method has a large sample capacity and measures 31.1, 15.7, and 7.43 nmol 8OHdG/L urine with variation coefficients of 8, 8 and 24% within series and 8, 11 and 23% between series. Normal healthy individuals were found to excrete 14.9 +/- 7.8 nmol 8OHdG/24 h, or 1.11 +/- 0.62 mumol 8OHdG per mol creatinine, in their urine, whereas increased levels of 8OHdG were found in 24 h collections from a variety of cancer patients, both in samples taken before onset of oncological therapy (1.84 +/- 1.12 mumol/mol creatinine, P < 0.01 versus healthy individuals) and after therapy onset (2.18 +/- 1.44 mumol/mol creatinine, P < 0.001 versus healthy individuals). Moreover, mean values of 8OHdG in random urinary samples from cancer patients were significantly higher than from healthy individuals (2.42 +/- 2.28 versus 1.19 +/- 0.48 mumol/mol creatinine, P < 0.001), both in samples taken before therapy onset (1.91 +/- 0.96, P < 0.001 versus healthy individuals) and after (2.57 +/- 2.46, P < 0.001 versus healthy individuals). High levels of urinary 8OHdG were found in patients subjected to whole body irradiation, and in patients receiving chemotherapy with various cytostatic agents. The potential use of the method for detecting increased urinary 8OHdG excretion and conditions associated with increased oxidative DNA damage in humans is discussed.

Cigarette smoke-induced DNA damage in cultured human lung cells: Role of hydroxyl radicals and endonuclease activation

Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.

Glucocorticoid treatment in ileal Crohn's disease: relief of symptoms but not of endoscopically viewed inflammation.

The effect of prednisolone (20-30 mg daily for six to nine weeks) was studied in eight patients with Crohns disease and recurrent, preanastomotic ileal inflammation, in respect of symptoms, endoscopic findings and phospholipase A2 activity in the ileal mucosa. The Harvey-Bradshaw Crohns disease activity index improved significantly, mainly because of reduced frequency of loose stools and diminution of abdominal pain. Endoscopy revealed no corresponding decrease in ileal inflammation, which in all cases persisted after treatment. The phospholipase A2 activity in the ileal mucosa was not altered by prednisolone. In two of three patients with concomitant colitis colonic inflammation improved. The study confirmed earlier reports of good symptomatic relief from glucocorticoid treatment in Crohns disease of the small bowel, but endoscopy suggests that this improvement was not the result of resolution of small intestinal mucosal inflammation.

Flavonoids as phospholipase A2 inhibitors: importance of their structure for selective inhibition of group II phospholipase A2.

The inhibitory effect of the plant flavonoid, rutin, on group I phospholipase A2 (PLA2-I) from porcine pancreas and Naja naja, and on group II phospholipase A2 (PLA2-II) from Vipera russelli and Crotalus atrox was investigated. Rutin efficiently inhibited PLA2-II from both Vipera russelli and Crotalus atrox but was only a weak inhibitor of PLA2-I from porcine pancreas and Naja naja. The lack of strong inhibition of pancreatic PLA2-I was not due to contaminating proteins in the enzyme preparation, since the same weak inhibition was obtained against pancreatic PLA2 purified to homogeneity as judged by two-dimensional gel electrophoresis. Rutin also efficiently inhibited human PLA2-II from synovial fluid but was only a weak inhibitor of human PLA2-I from pancreatic juice, suggesting that rutin is a selective PLA2-II inhibitor. A number of structurally similar flavonoids were tested for their ability to inhibit PLA2-II from Crotalus atrox and, for comparison, PLA2-I from porcine pancreas. The results obtained indicate that the hydroxyl group in 5-position as well as the double bond and the double-bonded oxygen in the oxane ring are all important for the overall ability of flavonoids to inhibit PLA2 activity, and that the hydroxyl groups in 3′- and 4′-position are required for selective inhibition of PLA2-II.

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.

Cigarette smoke-induced DNA-damage : role of hydroquinone and catechol in the formation of the oxidative DNA-adduct, 8-hydroxydeoxyguanosine

This study demonstrates the ability of cigarette smoke condensate to generate hydrogen peroxide and to hydroxylate deoxyguanosine (dG) residues in isolated DNA to 8-hydroxydeoxyguanosine (8-OHdG). Both the formation of hydrogen peroxide and that of 8-OHdG in DNA was significantly decreased when catalase or tyrosinase was added to the smoke condensates, and this also occurred when pure hydroquinone or catechol, two major constitutes in cigarette smoke, was used instead of smoke condensate. Moreover, pure hydroquinone and catechol both caused dose-dependent formation of hydrogen peroxide and 8-OHdG, and there was good correlation between the amounts of hydrogen peroxide and 8-OHdG formed. These findings suggest that (i) hydroquinone and catechol may be responsible for the ability of cigarette smoke to cause 8-OHdG formation in DNA, (ii) this oxidative DNA-damage is due to the action of hydroxyl radicals formed during dissociation of hydrogen peroxide and (iii) the hydrogen peroxide in cigarette smoke is generated via autooxidation of hydroquinone and catechol.

Passage of molecules through the wall of the gastrointestinal tract. I. A simple experimental model.

We describe here a simple experimental model for studying how the structural integrity of the intestinal wall is related to the transmural passage of molecules into the circulation. The model is based on the deposition of fluorescently labeled dextran in the intestine and its eventual recovery in the portal blood and mesenteric lymph. The fluorescent compound can be determined with sensitivity and ease by using fluorescence spectrometry. As judged from chromatography on Sephadex G-100, the compound was not degraded or otherwise chemically altered on its route of passage. The rate of passage was inversely proportional to the molecular size. The model may prove useful for studying factors that influence the transmission of macromolecules across the intestinal wall. Such a transmission probably underlies the pathogenesis of a variety of diseases.

Selective inhibition of group II phospholipase A2 by quercetin.

The influence of quercetin, chlorpromazine, aristolochic acid, and indomethacin on group I phospholipase A2 (PLA2) from porcine pancreas and on group II PLA2 fromVipera russelli was compared. Quercetin and chlorpromazine were found to inhibit PLA2 activity in lower concentrations (< 100μM), while aristolochic acid and indomethacin were inhibitory only in higher concentrations (> 100μM). The order of potency againstVipera PLA2 was: quercetin >chlorpromazin aristolochic acid > indomethacin, while the order of potency against pancreatic PLA2 was: chlorpromazine > aristolochic acid > indomethacin> quercetin. Thus, quercetin was a potent inhibitor towards group II PLA2 (IC50=2μM), but a very weak inhibitor against group I PLA2, with maximum 30% inhibition. Aristolochic acid and indomethacin were three to four times more potent towards group II PLA2 than towards group I PLA2, while chlorpromazine was equally potent towards the two PLA2 types. Quercetin and chlorpromazine were also tested against two PLA2 fractions purified from the plasma of septic shock patients; chlorpromazine was then equally potent towards the two PLA2 fractions, whereas quercetin was a potent inhibitor of only one of the two PLA2 fractions (IC50=4μM). Together, these results indicate that (1) different PLA2 inhibitors have different potency depending on which type of PLA2 they are used against, (2) quercetin selectively inhibits group II PLA2 and may therefore be used to discriminate between different PLA2 forms in biological materials, and (3) both PLA2 of group I and group II are present in septic shock plasma.

Phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia in rats.

The influence of various inflammatory inhibitors on the damaging effects of ischaemia in the small intestinal mucosa has been investigated. A rat experimental model was used, in which a ligated loop of the distal ileum was subjected to ischaemia and revascularisation and the ensuing mucosal damage assessed by lysosomal enzyme release and intestinal permeability measurements. The mucosal content of malondialdehyde - a lipid peroxidation product - and its activity of myeloperoxidase - a neutrophil granulocyte marker was also determined. In the absence of inhibitor, ischaemia and revascularisation caused increased mucosal permeability to sodium fluorescein, increased N-acetyl-beta-glucosaminidase release from the mucosa into the lumen, increased malondialdehyde content in the mucosa and increased myeloperoxidase activity in the mucosa. All these effects were inhibited by the phospholipase A2 inhibitors, quinacrine and nordihydroguaiaretic acid (NDGA), while the lipoxygenase inhibitor, BW755C, had no influence and the cyclooxygenase inhibitor, indomethacin, potentiated the increases in mucosal permeability and N-acetyl-glucosaminidase release. BN 52021, a specific platelet activating factor antagonist, did not influence the myeloperoxidase activity, but it decreased the formation of malondialdehyde and the increases in mucosal permeability and N-acetyl-beta-glucosaminidase release, although not to the same extent as quinacrine and NDGA. These findings indicate that phospholipase A2 inhibition prevents mucosal damage associated with small intestinal ischaemia and suggest that at least part of the ischaemic damage is mediated by products of phospholipase A2 activity that are not arachidonic acid metabolites.