Neville A. Punchard

The mode of action of the aminosalicylates in inflammatory bowel disease

Sulphasalazine and other 5‐aminosalicylic acid (5‐ASA)‐containing drugs are used in the treatment of acute inflammatory bowel disease and in the maintenance of clinical remission. Despite their use for over 50 years, the mechanism of action of this class of drugs remains uncertain, although a number of possibilities are discussed in this review. It seems likely that the aminosalicylates are important free radical scavengers, can reduce leukotriene production and can inhibit the cellular release of interleukin‐1, all of which are likely to be important in reducing the acute inflammatory response in inflammatory bowel disease, The effects of these drugs on prostaglandin production are more contentious, but it appears that 10‐5 to 10‐4 M concentrations stimulate production of prostaglandins which may be cytoprotective, while higher doses of these drugs inhibit prostaglandin production. The aminosalicylates may maintain remission in inflammatory bowel disease by preventing leucocyte recruitment into the bowel wall. The drugs inhibit the chemotactic response to leukotriene B4, reduce the synthesis of platelet activating factor and also inhibit leucocyte adhesion molecule upregulation.

A randomized controlled study of evening primrose oil and fish oil in ulcerative colitis

In a placebo‐controlled study, 43 patients with stable ulcerative colitis were randomized to receive either MaxEPA (n= 16), super evening primrose oil (n= 19), or olive oil as placebo (n= 8) for 6 months, in addition to their usual treatment. Treatment with MaxEPA increased red‐cell membrane concentrations of eicosapentaenoic acid (EPA) at 3 months by three‐fold and at 6 months by four‐fold (both P < 0.01), and doubled docosahexaenoic acid (DHA) levels at 6 months (P < 0.05). Treatment with super evening primrose oil increased red‐cell membrane concentrations of dihomogamma‐linolenic acid (DGLA) by 40% at 6 months (P < 0.05), whilst treatment with placebo reduced levels of DGLA and DHA at 6 months (both P < 0.05). Clinical outcome was assessed by patient diary cards, sigmoidoscopy and histology of rectal biopsy specimens. Super evening primrose oil significantly improved stool consistency compared to MaxEPA and placebo at 6 months, and this difference was maintained 3 months after treatment was discontinued (P < 0.05). There was however, no difference in stool frequency, rectal bleeding, disease relapse, sigmoidoscopic appearance or rectal histology in the three treatment groups. Despite manipulation of cell‐membrane fatty acids, fish oils do not exert a therapeutic effect in ulcerative colitis, while evening primrose oil may be of some benefit.

Glucocorticoid-mediated transrepression is regulated by histone acetylation and DNA methylation.

Glucocorticoids are highly effective in controlling chronic inflammatory diseases by inhibiting the expression of cytokines and chemokines. Glucocorticoids act through binding of their receptor resulting to inhibition of transcription factors such as nuclear factor kappa B (NF-kappa B). This may occur via the transcription integrator protein, CREB binding protein (CBP), which has intrinsic histone acetylase (HAT) activity. Interleukin (IL)-1 beta caused a significant increase in NF-kappa B-mediated granulocyte/macrophage colony stimulating factor (GM-CSF) release, which was inhibited by the glucocorticoid mometasone furoate (MF) (EC(50)=2 x 10(-11) M). This effect was inhibited by CBP over-expression. The role of histone acetylation and DNA methylation in the transcription of GM-CSF was indicated by trichostatin A (TSA), an inhibitor of histone deacetylases, and 5-azacytidine (5-aza), a DNA methylase inhibitor, to increase GM-CSF expression partially blocking glucocorticoid inhibition of IL-1 beta-stimulated GM-CSF release. These data suggest that the mechanism of glucocorticoid action in suppressing interleukin-1 beta-stimulated GM-CSF release in A549 cells may involve modulation of CBP-mediated histone-acetylase activity and DNA methylation.

Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCε and JAK2 in RAW macrophages

BackgroundNuclear factor kappa B (NF-κB) has been shown to play an important role in regulating the expression of many genes involved in cell survival, immunity and in the inflammatory processes. NF-κB activation upregulates inducible nitric oxide synthase leading to enhanced nitric oxide production during an inflammatory response. NF-κB activation is regulated by distinct kinase pathways independent of inhibitor of κB kinase (IKK). Here, we examine the role of protein kinase C isoforms and janus activated kinase 2 (JAK2) activation in NF-κB activation and LPS-stimulated NO production.MethodsMurine RAW 264.7 macrophages were treated with lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA) and a combination of LPS and PMA in the presence or absence of various inhibitors of PKC isoforms and JAK2. Nuclear translocation of the NF-κB p65 subunit, was assessed by Western blot analysis whilst NO levels were assessed by Greiss assay.ResultsLPS-stimulated NO production was attenuated by PMA whilst PMA alone did not affect NO release. These effects were associated with changes in p65 nuclear translocation. The PKCα, β, γ, δ and ζ inhibitor Gö 6983 (Go) had no effect on LPS-induced NO release. In contrast, Bisindolymalemide I (Bis), a PKC α, βI, βII, γ, δ and ε isoform inhibitors completely inhibited LPS-stimulated NO production without affecting p65 nuclear translocation. Furthermore, a partial inhibitory effect on LPS-induced NO release was seen with the JAK2 inhibitor AG-490 and the p38 MAPK inhibitor SB 203850.ConclusionThe results further define the role of NF-κB in LPS stimulated NO production in RAW macrophages. The data support a function for PKCε, JAK2 and p38 MAPK in NF-κB activation following p65 nuclear import.

Activation of nuclear factor kappa B in Crohn's disease

Abstract.Objectives and Design: The location and degree of activation of nuclear factor kappa (NFκB), a primary transcription factor that plays a regulating role in immune and inflammatory responses, was determined in Crohns disease using full thickness specimens of bowel collected at surgery.¶Materials and Methods: Resected specimens of inflamed and non-inflamed bowel were collected from thirteen patients with Crohns disease and non-inflamed bowel from eleven control subjects. Prepared frozen sections were immunostained using a monoclonal antibody to the activated form of the p65 subunit of NFκB and the number of positive staining cells counted using a Lennox graticule.¶Results: The number of cells positive for activated NFκB was significantly increased (p = 0.001) in all layers of inflamed Crohns disease bowel, compared to non-inflamed bowel from controls. There was also a significant increase (p = 0.009) in the number of positive cells, when compared to non-inflamed bowel from control subjects, in the submucosa of non-inflamed areas of Crohns disease bowel. Cells positive for activated NFκB were provisionally identified by morphological criteria as mostly macrophages with some lymphocytes. There was no activation in endothelia.¶Conclusion: NFκB is activated within large mononuclear cells in all layers of inflamed areas of the bowel in Crohns disease and may represent key events in the inflammatory process. Increased activation in the submucosa of non-inflamed Crohns disease bowel provides further evidence of early immunological activation in macroscopically and microscopically uninvolved areas and an underlying abnormal immune system in Crohns disease.

Differential patterns of histone acetylation in inflammatory bowel diseases

Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohns disease (CD) to study the expression of acetylated histones (H) 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K) 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyers patches (PP) in dextran sulfate sodium (DSS)-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

Red cell lipid peroxidation and antioxidant enzymes in iron deficiency

Abstract:  Whether iron deficient RBC in humans have a reduced, or an increased, susceptibility to lipid peroxidation was studied in the iron deficiency states of primary proliferative polycythaemia and iron deficiency anaemia and related to changes in the activities of iron‐dependent and non‐iron dependent antioxidant enzymes. Susceptibility of RBCs to lipid peroxidation was increased when expressed per g Hb. However, this was a result of the low RBC Hb giving an increased membrane lipid: Hb ratio in the incubations. Results were normal when expressed either per cell, or per ml, RBC. Glutathione reductase was normal. Increased RBC superoxide dismutase activity in iron deficiency may be explained by the younger RBC population and reductions in glutathione peroxidase and catalase activities by the microcytic hypochromic changes and the lack of availability of iron, respectively. There is no evidence of an increased susceptibility of RBC to lipid peroxidation in iron deficiency.

Inhibition of leucocyte adhesion molecule upregulation by tumor necrosis factor alpha: a novel mechanism of action of sulphasalazine.

The effects of the cytokine tumour necrosis factor alpha and the calcium ionophore A23187 upon CD11a, CD11b, CD11c and CD18 leucocyte membrane expression was analysed in whole blood using monoclonal antibodies and flow cytometry. Both agents significantly increased the density of CD11b/CD18 membrane expression on monocytes and granulocytes, but had no effects on adhesion molecule expression on lymphocytes. The effects of sulphasalazine, 5-aminosalicylic acid (5-ASA) and sulphapyridine upon adhesion molecule upregulation were then examined; 10(-3) and 10(-4) M sulphasalazine and 5-ASA significantly reduced tumour necrosis factor alpha induced CD11b/CD18 upregulation on monocytes and granulocytes but had no effects upon A23187 mediated upregulation. Sulphapyridine was inactive. These results suggest that sulphasalazine and 5-ASA may interfere with mechanisms of leucocyte recruitment in inflammatory bowel disease.

The Journal of Inflammation

Welcome to the Journal of Inflammation, the first open-access, peer-reviewed, online journal to focus on all aspects of the study of inflammation and inflammatory conditions. While research into inflammation has resulted in great progress in the latter half of the 20th century, the rate of progress is rapidly accelerating. Thus there is a need for a vehicle through which this very diverse research can be made readily available to the scientific community. The Journal of Inflammation, a peer reviewed journal, provides the ideal vehicle for such rapid dissemination of information. The Journal of Inflammation covers the full range of underlying cellular and molecular mechanisms involved, not only in the production of the inflammatory responses but, more importantly in clinical terms, in the healing process as well. This includes molecular, cellular, animal and clinical studies related to the study of inflammatory conditions and responses, and all related aspects of pharmacology, such as anti-inflammatory drug development, trials and therapeutic developments, etc. All articles published in the Journal of Inflammation are immediately listed in PubMed, and access to published articles is universal and free through the internet.

Lipid peroxidation in rats chronically fed ethanol.

Chronic alcohol consumption induces cytochrome P450IIE1, enabling habitual abusers to consume far greater quantities of alcohol than normal subjects. This pathway of metabolism leads to the production of free radical species, which cause tissue damage through peroxidation of cell membranes. Groups of Wistar rats of equal male: female ratio (n = 24) were fed alcohol by gavage twice daily to achieve a dosage of 15 g/kg body weight. Mean peak blood alcohol concentrations of 186 mg% were produced in males and 156 mg% in females. The animals were allowed free access to standard laboratory chow and water. Control animals were pair-fed to the alcoholic group and fed isocaloric glucose by gavage. Groups of animals were killed between 9 and 11 am on consecutive mornings, after nocturnal feeding, since it has previously been shown that fasting rapidly depletes hepatic glutathione concentrations. Hepatic glutathione was measured by a spectrophotometric enzymatic recycling procedure. As a marker of lipid peroxidation hepatic malonaldehyde (MDA) was measured by high performance liquid chromatography. Hepatic MDA was increased in the alcoholic group (p < 0.001), as was total hepatic glutathione (p < 0.0001). Plasma concentrations of alpha-tocopherol were increased in the alcoholic group, but ascorbic acid and superoxide dismutase values were not affected. No sex differences were detected. The increased MDA production in the alcohol group is strong evidence that lipid peroxidation is a mechanism of alcoholic tissue damage. The rise in hepatic glutathione may be an adaptive response to free radical production that protects the rat against tissue damage.