Richard A. Mumford

Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease

BACKGROUND & AIMS Inducible nitric oxide synthase (iNOS) is generated in several cell types by treatment with lipopolysaccharides or cytokines. Earlier studies suggested that ulcerative colitis is associated with increased NO produced by iNOS; however, the cellular source of the NO synthesis was not identified. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide to produce peroxynitrite, which reacts with tyrosine to form nitrotyrosine in cellular proteins. METHODS Using immunoperoxidase microscopy with a new monospecific human iNOS antibody (NO-53), the cellular distribution of iNOS and nitrotyrosine was examined using human colonic mucosa from normal bowel, ulcerative colitis, Crohns disease, and diverticulitis. RESULTS Intense focal iNOS labeling was localized to the inflamed colonic epithelium in ulcerative colitis, Crohns disease, and diverticulitis but was not detectable in the uninflamed epithelium. Nitrotyrosine labeling was also observed in the inflamed colonic epithelium and was associated with nearby iNOS staining; nitrotyrosine was undetectable in normal mucosal epithelium. iNOS and nitrotyrosine were also detected in lamina propria mononuclear cells and neutrophils. CONCLUSIONS These findings suggest that iNOS is induced in the inflamed human colonic epithelium and is associated with the formation of peroxynitrite and the nitration of cellular proteins.

VDIPEN, a metalloproteinase-generated neoepitope, is induced and immunolocalized in articular cartilage during inflammatory arthritis.

The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.

The Gaucher mouse.

Abstract A model of Gauchers disease was produced through the administration of conduritol B-expoxide. Tissue levels of glucosylceramide were elevated in the experimental animals. The activity of β-glucosidase in homogenates of brain, liver, and spleen was reduced 93% in the treated animals. Six other lysosomal hydrolases measured were uneffected.

The discovery of sulfonylated dipeptides as Potent VLA-4 antagonists

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.

The fibrinogen cleavage product A alpha-Val(360), a specific marker of neutrophil elastase activity in vivo

Background Alpha-1-antitrypsin (A1AT) deficiency is the only recognised genetic risk factor for chronic obstructive pulmonary disease (COPD), a leading cause of morbidity and mortality worldwide. Since A1AT is the major inhibitor of neutrophil elastase (NE), this enzyme has become widely implicated in the pathogenesis of COPD in general; however, there is currently no specific biomarker for its pre-inhibition activity. Such a biomarker should be a measure of elastase-specific COPD disease activity with the potential to assess early targeted therapeutic intervention, in contrast to traditional and non-specific disease severity markers such as forced expiratory volume in 1 s. Methods In pilot studies, plasma Aα-Val360 and markers of neutrophil activation were measured in 95 subjects with a range of A1AT concentrations. Aα-Val360 and sputum elastase activity were also measured in a further seven PiZ A1AT-deficient subjects over the course of an acute exacerbation. Finally, Aα-Val360 was measured in plasma from subjects randomised to receive A1AT replacement or placebo in the EXACTLE trial. Results The plasma concentrations of Aα-Val360 and A1AT related exponentially, consistent with previous theoretical and in vitro experimental data. L-233 (an intracellular NE inhibitor) blocked generation of Aα-Val360 and subsequent A1AT/NE complex formation. Aα-Val360 was related to the spirometric severity of lung disease in A1AT deficiency, to sputum elastase activity in acute exacerbations and was decreased in subjects receiving A1AT replacement therapy (while remaining constant in those receiving placebo). Conclusions Aα-Val360 represents the first specific footprint of pre-inhibition NE activity and is a potential biomarker of disease activity and progression in subjects with elastase-dependent COPD. Trial registration The EXACTLE study was registered in ClinicalTrials.gov as ‘Antitrypsin (AAT) to Treat Emphysema in AAT-Deficient Patients’; ClinicalTrials.gov Identifier: NCT00263887.

N-Aryl 2,6-Dimethoxybiphenylalanine Analogues as VLA-4 Antagonists

A series of N-arylated phenylalanine derivatives has been synthesized and has been shown to be potent inhibitors of the integrin VLA-4. N-phenyl and N-heteroaryl derivatives with hydrogen bond acceptors in the meta position demonstrated low nanomolar activity against VLA-4.

PMN elastases: A comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Deficiency of glucosylsphingosine: -Glucosidase in Gaucher disease

Abstract A deficiency in the activity of glucosylsphingosine: β-glucosidase has been observed in Gauchers spleen tissue and skin fibroblasts. Preliminary studies indicated the presence of a material similar to glucosylsphingosine in Gauchers spleen while such a material was not detectable in normal and other pathological control spleen tissue. This is the first report of the natural occurrence of a psychosine-like material in a mammalian tissue.

Aα-Val360: a marker of neutrophil elastase and COPD disease activity

Forced expiratory volume in 1 s is currently the most widely used marker of chronic obstructive pulmonary disease (COPD) severity; however, it is a poor surrogate of the emphysematous component and the underlying pathophysiological mechanism, and therefore new markers are urgently needed. Neutrophil elastase (NE) is likely to play a key pathophysiological role in COPD and the current study explores a marker of NE activity as a potential indicator of COPD disease activity. A&agr;-Val360 was measured in 81 subjects with a clinical diagnosis of COPD, both in the stable state and at presentation with an acute exacerbation, and comparisons were made using lung function tests and computed tomography imaging. The relationship of A&agr;-Val360 with disease progression was also assessed in 40 of the subjects over a 4-yr period. Baseline A&agr;-Val360 related to physiological and radiological markers of disease severity, was higher at presentation with an acute exacerbation than in the stable state and (at least partly) related to disease progression over the subsequent 4 yrs. We demonstrate that A&agr;-Val360 is a marker of cross-sectional COPD disease severity and possibly disease progression, and represents a new concept of specific biomarkers. This study therefore reports the first in vivo data to support the pathophysiological role of NE in COPD.

Protease activities present in wheat germ and rabbit reticulocyte lysates

Abstract Rabbit reticulocyte lysates and wheat germ lysates were found to contain significant neutral protease activity when assayed against the highly sensitive 7-amino-4-methylcoumarin (AMC) peptide substrates Phe-AMC, succinyl-Ala-Ala-Phe-AMC and t-boc-Ala-Ala-Pro-Ala-AMC (substrates for aminopeptidase, chymotrypsin and elastase-like enzymes, respectively). Additionally, wheat germ lysates contain a trypsin-like activity when assayed against CBZ-Gly-Gly-Arg-AMC and a post-proline cleaving activity which hydrolyzed the Pro-Ala bond of t-boc-Ala-Ala-Pro-Ala-AMC.