Screening and monitoring coeliac disease: multicentre trial of a new serum antibody test kit.

A multicentre trial was conducted to evaluate a new test for anti-gliadin antibodies (AGA) in serum (Coeliac Screening Kit, CSK, Medical Innovations Limited, Artarmon, NSW, Australia). The test showed excellent reproducibility for both anti-gliadin IgA and IgG detection. The average intraassay coefficient of variation (CV) was 3.0% for IgA and 2.4% for IgG (n = 6), while the average interassay CV was 6.4% for IgA and 4.3% for IgG (n = 3). By defining a positive test as both IgA and IgG elevated, a sensitivity of 93% in untreated coeliacs (n = 75) was observed. The corresponding specificities in healthy adults (n = 130) and healthy children (n = 77) were > 99% and 100% respectively, while in patients with other gastrointestinal disorders (disease controls) the specificity was 94% (n = 129). The test was also useful in monitoring patients, with anti-gliadin IgA and IgG falling for up to a year after commencing a gluten-free diet (GFD) (12 adults). In some patients however, antibody levels did not reach the normal cutpoint after many months on a GFD, which may reflect the patients' poor adherence to their gluten free diet. The test was superior to the Pharmacia anti-gliadin ELISA, and should be useful as an aid to the diagnosis of coeliac disease, as well as in the follow-up of treated patients.

Minactivin expression in human monocyte and macrophage populations.

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.

Novel properties of human monocyte plasminogen activator.

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.

Minactivin: a human monocyte product which specifically inactivates urokinase-type plasminogen activators.

Culture supernatants from monolayers of human peripheral monocytes strongly inhibited colorimetric assays of urokinase in which plasmin was measured by esterolysis. This inhibitory activity of monocyte culture supernatant was enhanced after culture with muramyl dipeptide. Inhibition was specific for plasminogen activators of Mr 52 000 and 36 000, as shown by three methods: (1) inhibition of plasminogen-dependent fibrinolysis; (2) inhibition at the level of plasminogen activation in a colorimetric assay; (3) the irreversible loss of plasminogen-activating activity, as evidenced by electrophoresis, after preincubation with culture media. The factor responsible for this inactivation (which we propose to call minactivin) had an apparent Mr of 66 000 on Sephacryl S300 gel chromatography and interacted with enzyme in a biphasic manner: a rapid partial inhibition (reversible by sodium dodecyl sulphate) was followed by slow inactivation (irreversible by sodium dodecyl sulphate). It is proposed that secretion of minactivin by monocytes may contribute to regulation of extracellular proteolysis at sites of tissue injury.

Isolation and preliminary characterization of human intestinal macrophages.

Methods for the isolation and culture of macrophages from normal human intestine are described. After disaggregation using sequential treatments of dithiothreitol, ethylenediaminetetraacetate, collagenase, and deoxyribonuclease, Percoll density gradients (1.064 SG) produced single cell suspensions from which macrophages were readily purified by adherence to plastic. Macrophages were characterized by morphology, phagocytosis, cytoplasmic staining for nonspecific esterase, presence of membrane Fc receptors, Ia-like antigens, and lysozyme synthesis and secretion. In 10 separate experiments, recovery of viable mononuclear cells was 2.9 +/- 0.6 x 10(6) cells/g of mucosa. Thirty percent of these cells were phagocytic. After adherence to plastic, the macrophage recovery was 1.05 +/- 0.2 x 10(6) cells/g mucosa and 90% +/- 0.4% of the adherent cells in the monolayer were phagocytic. Fifty-five percent of the adherent cells showed Fc receptors for immunoglobulin G, while 94% expressed the Ia-like antigen on their membrane. The successful isolation and culture of human intestinal macrophages in large numbers will allow detailed study of their role in the mucosal immune response in health and disease.