Characterisation of anti-neutrophil cytoplasmic antibody target antigens using electrophoresis and western blotting techniques.

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies which react with components of phagocytic cells, and are associated with vasculitis and other idiopathic inflammatory disorders. However, the antigenic targets of many of these autoantibodies have not been defined yet. In this study, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) were evaluated for characterising the antigenic specificity of unidentified ANCA. The uncharacterised sera included those from patients with ulcerative colitis (n = 21), Crohn's disease (n = 5), cystic fibrosis (n = 16) and sarcoidosis (n = 2). In addition, sera from patients with antibodies to the phagocytic enzymes proteinase 3 (PR3) (n = 11) and myeloperoxidase (MPO) (n = 5) were also included. The sub-cellular localisation of antigens was determined by testing sera against crude neutrophil extract and sub-cellular fractions consisting of azurophilic granules, specific granules and cytosolic, fractions using enzyme-linked immunosorbent assays (ELISAs). All sera reacted with the crude and azurophilic granule extracts. The native system of IEF followed by capillary immunoblotting successfully detected anti-PR3 and anti-MPO in azurophilic granule extracts. In contrast, SDS-PAGE Western blotting failed to detect any reactivity, either to PR3 or MPO, in the crude extract or azurophilic granule extract. However, the antibody specificity of patient sera with uncharacterised autoantibodies could not be detected by IEF/capillary immunoblotting or SDS-PAGE. This study showed that the sub-cellular azurophilic granules are the antigenic target of a variety of uncharacterised ANCA. It also showed that IEF characterised both anti-PR3 and anti-MPO but failed to detect other forms of ANCA. In contrast, the majority of common ANCA were not detected by SDS-PAGE.     

Endoscopic alternatives in the management of colonic strictures

A 10-year review of our experience with all patients with symptoms of colonic narrowing (n = 61) revealed 14 patients who were treated endoscopically. The site of narrowing was the sigmoid colon in 12 patients and the rectum in two patients. The strictures occurred after anastomosis in seven patients, with carcinoma in four patients, and with inflammatory disease, external compression, and idiopathy in one patient each. Although combinations of endoscopic techniques were occasionally used, the predominant method responsible for successful management of the narrowing was bouginage in four patients, endoscopy with a prototype dilating endoscope in four patients, balloon dilatation in three patients, and electrocautery and laser surgery in one patient each. There were no perforations or bleeding complications. Repeated treatments were usually needed. As less invasive methods evolve to treat colonic narrowing, appropriate matching of available techniques with the underlying disease becomes easier. We have found that dilation with a bougie, balloon, or a prototype dilating endoscope can provide especially beneficial results when used on patients with strictures resulting from inflammatory disease or external compression. Cutting and ablating tools such as the electrocautery and laser tools are more suited for management of strictures that result from carcinoma and anastomotic webs. Appropriate matching of endoscopic technique to underlying colonic pathology will allow increasingly successful and safer management of colonic narrowing without operation.