T. T. Macdonald

Tumour necrosis factor alpha in stool as a marker of intestinal inflammation

Measurement of disease activity in patients with inflammatory bowel disease is difficult. The best available methods are complex and time consuming, but it may be possible to use tumour necrosis factor alpha (TNF alpha) concentration in stool as a marker of disease activity. We measured TNF alpha concentrations in stool samples from normal children, infants with diarrhoea, and children with inflammatory bowel disease in active and inactive phases of the disease. In 10 normal children and 14 children with diarrhoea, median stool TNF alpha concentrations were 58 and 45 pg/g stool, respectively. Compared with diarrhoeal controls, stool TNF alpha concentrations were significantly increased in children with active Crohns disease (n = 13, median 994 pg/g, p less than 0.0002) and active ulcerative colitis (n = 4, range 276-5982 pg/g, p less than 0.003). In patients with inactive disease, either as a result of surgery or treatment with steroids, the concentration of stool TNF alpha fell to those of controls. Measurement of stool TNF alpha concentrations may provide a simple way to monitor disease activity in inflammatory bowel disease.

Tumour necrosis factor-alpha and interferon-gamma production measured at the single cell level in normal and inflamed human intestine

The spot‐ELISA technique has been used to enumerate the frequency of cells secreting tumor necrosis factor‐alpha (TNF‐α) and interferon‐γ (IFN‐γ), isolated from biopsies of normal intestine and from biopsies of children with inflammatory bowel disease. TNF‐α production was undetectable in six out of 12 biopsies from normal intestine and in the other six biopsies it ranged from 60 to 580 TNF‐α‐secreting cells/106 isolated intestinal cells. In contrast, cells isolated from biopsies of children with Crohns disease (n= 9) all showed elevated frequencies of TNF‐á‐secreting cells (500–12 000 secreting cells/106 cells). In ulcerative colitis, four out of eight children had increased production of TNF‐α and in children with indeterminate colitis two out of three had elevated levels. There was no correlation between plasma TNF‐α levels and the number of intestinal cells secreting TNF‐α. In controls and all groups of patients IFN‐γ‐secreting cells were uncommon. These results suggest that TNF‐α is an important mediator of inflammation in the human gut, and, furthermore, may play a role in the growth failure frequently seen in children with inflammatory bowel disease.

Measles vaccination as a risk factor for inflammatory bowel disease.

Measles virus may persist in intestinal tissue, particularly that affected by Crohns disease, and early exposure to measles may be a risk factor for the development of Crohns disease. Crohns disease and ulcerative colitis occur in the same families and may share a common aetiology. In view of the rising incidence of inflammatory bowel disease (Crohns disease and ulcerative colitis), we examined the impact of measles vaccination upon these conditions. Prevalences of Crohns disease, ulcerative colitis, coeliac disease, and peptic ulceration were determined in 3545 people who had received live measles vaccine in 1964 as part of a measles vaccine trial. A longitudinal birth cohort of 11,407 subjects was one unvaccinated comparison cohort, and 2541 partners of those vaccinated was another. Compared with the birth cohort, the relative risk of developing Crohns disease in the vaccinated group was 3.01 (95% CI 1.45-6.23) and of developing ulcerative colitis was 2.53 (1.15-5.58). There was no significant difference between these two groups in coeliac disease prevalence. Increased prevalence of inflammatory bowel disease, but not coeliac disease or peptic ulceration, was found in the vaccinated cohort compared with their partners. These findings suggest that measles virus may play a part in the development not only of Crohns disease but also of ulcerative colitis.

High endothelin-1 immunoreactivity in Crohn's disease and ulcerative colitis

Both immunological hypersensitivity and vascular abnormalities have been implicated in the pathogenesis of inflammatory bowel disease. In an attempt to link the two hypotheses, we sought evidence of local production of endothelin-1, a potent vasoconstrictor, in patients with Crohns disease and ulcerative colitis. An immunohistochemical method was used to detect endothelin-1 in tissue samples from sixteen Crohns disease patients, nine ulcerative colitis patients, and thirteen controls. In the controls, positively staining cells were infrequent in both lamina propria (mean 0.9% of total cells, 95% confidence interval 0.1-1.7%) and submucosa (2.3%, 0.4-4.1%). The percentage of endothelin-immunoreactive cells was significantly higher in the two disease groups than in the controls. Among the Crohns disease patients, there were more immunoreactive cells in the submucosa than in the lamina propria (19.1%, 15.2-22.1% vs 12.3%, 8.1-16.5%; p less than 0.001), whereas the converse was true for the ulcerative colitis group (8.6%, 1.1-16.1% vs 24.4%, 14.1-34.6%; p less than 0.001). Immunoreactive macrophage aggregates around submucosal blood vessels were common in samples from Crohns disease patients. Endothelin concentrations, measured by radioimmunoassay, in supernatants of homogenised tissue samples were significantly higher in Crohns disease and ulcerative colitis than in controls. We suggest that local endothelin production by inflammatory cells may contribute to vasculitis in chronic inflammatory bowel disease by inducing intestinal ischaemia through vasoconstriction.

Disruption of sulphated glycosaminoglycans in intestinal inflammation

We have studied the distribution and nature of sulphated glycosaminoglycans (GAGs) within normal and inflamed intestine. There is increasing evidence that these negatively charged polysaccharides, which both regulate the ability of albumin to leave the vasculature and inhibit thrombosis, may be affected by inflammatory cells and their products. We obtained samples of freshly resected intestinal tissue from eight controls, eleven patients with Crohns disease, and six with ulcerative colitis. Sulphated GAGs were detected by means of a gold-conjugated poly-L-lysine probe, and the tissue density of anionic sites was assessed semiquantitatively by means of a Lennox graticule. In normal intestine there was staining in the vascular endothelium and the subepithelial basal lamina and throughout the extracellular matrix of the lamina propria and submucosa. Tissue from the patients with inflammatory bowel disease showed inflammation macroscopically and on histology. There were profound abnormalities of extracellular matrix GAGs, limited to the mucosa in ulcerative colitis and greatest in the submucosa in Crohns disease. There was also substantial loss of GAGs from the subepithelial basal lamina in both disorders and from the vascular endothelium in submucosa in Crohns disease. The extent of local GAG disruption was associated with the distribution of macrophages immunoreactive for tumour necrosis factor alpha and the activation marker RM 3/1. We suggest that inflammatory disruption of vascular and connective tissue GAGs may be an important pathogenetic mechanism, contributing to the leakage of protein and fluid, thrombosis, and tissue remodelling seen in inflammatory bowel disease.

Gamma/delta T cells and the diagnosis of coeliac disease

Gamma/delta T cells are increased in the gut epithelium of patients with coeliac disease compared with normal controls. The aim of this study was to determine whether the increase in yd intraepithelial lymphocytes (IEL) is specific for coeliac disease, in which case it could be of diagnostic importance. Biopsies were obtained from children with no intestinal disease, coeliac disease, cow‐milk‐sensitive enteropathy/post‐enteritis syndrome (CMSE/PES) and miscellaneous other enteropathies (n = 67). Intraepithelial CD3+ and γδ T cells were identified in frozen sections using peroxidase immunohisto‐chemistry. In normal biopsies there were 0‐7 γδ IEL/100 cells in the epithelium. In untreated coeliac patients this increased to 9‐22 γδ IEL/100 cells in the epithelium (P=0.000004). Of 27 patients with morphologic intestinal damage which was not due to coeliac disease, four with CMSE/PES had γδ IEL/100 cells in the epithelium in the same range as the patients with coeliac disease. Of these, two had high densities of CD3+ IEL in the epithelium and were indistinguishable from patients with untreated coeliac disease. The other two could be excluded as possible coeliacs because their CD3f IEL/100 epithelial cells were in the normal range. Thus an increase in γδ IEL is not specific for coeliac disease. However, enumeration of both of γδ IEL and CD3+ IEL densities will be useful in the exclusion of coeliac disease as a diagnosis in some children.

Intestinal nematode infections in children : the pathophysiological price paid

The mechanism by which small animals such as rodents resist or eliminate nematode parasites requires mucosal inflammation as the final effector of the immune response. The resulting freedom from chronic infection may be worth the price of short-term illness. Putative vaccines which attempt to enhance the natural effect will have to take into account the inflammatory cost to the host. Human helminthiases involve a more stable equilibrium between host and parasite. The medical literature on hookworm disease and clinical ascariasis describes, for the former, some chronic inflammatory effects correlated with worm burden, but for the latter a less quantified or predictable set of detrimental effects. We describe a current, systematic study of the inflammatory response to whipworm infection, in which anaemia, growth retardation and intestinal leakiness are viewed as predictable consequences related to infection intensity. There is evidence for the absence of cell-mediated immunopathology. However, a specific, IgE-mediated local anaphylaxis may, at least partly, mediate the deleterious effects. Increased numbers of mucosal macrophages may also contribute to the chronic, systemic effects through their output of cytokines. Similar attempts to show the mechanisms of pathogenesis and quantify the effects of hookworm disease should be undertaken.

Immediate hypersensitivity in colon of children with chronic Trichuris trichiura dysentery

There are few data on mucosal immune responses to intestinal helminths in human beings, especially those involving the IgE system, which is thought to be important in parasite expulsion. We sought evidence of an immediate hypersensitivity reaction in the colon of children with chronic dysentery due to Trichuris trichiura. 28 children with Trichuris dysentery syndrome (TDS) were compared with 16 control children (with no TDS or worms visible on colonoscopy). All children were aged 1-11 years. Rectal biopsy samples were taken before and after expulsion of the worms by means of mebendazole treatment. Children with TDS had significantly greater numbers than controls of mast cells (mean [SD] 10.9 [1.3] vs 3.9 [0.6]% of all cells; p less than 0.0003) and of cells with surface IgE (median [range] 11.1 [7.5-11.6] vs 1.0 [0-1.5]%; p less than 0.001) in the subepithelial region of the mucosa. On electronmicroscopy, degranulating mast cells were prominent in parasitised children. In culture, rectal biopsy samples from parasitised children showed high rates of spontaneous histamine release, but only low rates of antigen-specific release. After treatment, spontaneous histamine release was significantly reduced and antigen-specific histamine release could be provoked. Thus, an IgE-mediated immune mucosal response to a helminth infection does occur in human beings but is not sufficient to cause appreciable parasite expulsion.

3. Mucosal macrophages and cytokine production in the colon of children with Trichuris trichiura dysentery

Mucosal macrophages and accessory cells have been studied by immunohistochemistry in the lamina propria of the colon of children with Trichuris trichiura dysentery syndrome (TDS). No difference was found in the numbers of cells recognized by the monoclonal antibodies CD11c, CD68, or RFD7 between TDS children and local controls. However, large numbers of cells were recognized by an antibody against calprotectin (an anti-bacterial glycoprotein found in tissue infiltrating-monocytes) in TDS colonic mucosa, but few in control colon. Large numbers of cells containing tumour necrosis factor alpha (TNF alpha) were also seen in TDS mucosa; cells isolated from TDS mucosa secreted more TNF alpha than cells from control mucosa; and children with TDS had high levels of circulating TNF alpha. Non-specific macrophage-mediated inflammation and local cytokine production may therefore play a role in the pathogenesis of TDS.

Urinary growth hormone in growth-impaired children with chronic inflammatory bowel disease.

&NA; Growth impairment of undefined aetiology occurs in ˜30% of children with chronic inflammatory bowel disease. We measured urinary growth hormone concentrations in 36 children with chronic inflammatory bowel disease and 51 normal controls. The median urinary concentration of growth hormone in the stunted children with chronic inflammatory bowel disease was 15.8 ng/g creatinine (range 4.3‐32.6), compared with 11.7 ng/g creatinine (range 4.1‐35.9) in those with normal growth. The difference was statistically not significant (p = 0.15). Moreover, there was no significant difference between the patients and the control group. One stunted patient had a urinary growth hormone (UGH) concentration below the normal range, whereas four patients with normal height were also below the normal range. Four of these five patients (80%) were on corticosteroid treatment at the time of urinary collection, whereas only 26% of the patients with normal UGH were also on corticosteroid treatment. We conclude that growth retardation in children with chronic inflammatory bowel disease is probably not related to growth hormone deficiency but must involve other mechanisms.