Duncan A.F. Robertson

Osteoporosis in treated adult coeliac disease.

Forty five women and 10 men with coeliac disease diagnosed in adult life, who were already on a gluten free diet, had serial bone mineral density measurements at the lumbar spine and femoral neck over 12 months. Osteoporosis, defined as a bone mineral density (BMD) < or = 2 SD below the normal peak bone mass was found in 50% of male and 47% of female coeliac patients. Patients with a BMD < or = 2 SD below age and sex matched normal subjects, had a significantly lower body mass index (21.3 kg.m-2 compared with 25.2 kg.m-2, p < 0.02 Wilcoxon rank sum test) and lower average daily calcium intake (860 mg/day compared with 1054 mg/day, p < 0.05 Wilcoxon rank sum test) than patients with normal bone mineral density. In postmenopausal women with coeliac disease there was a strong correlation between the age at menopause and BMD at both the lumbar spine (r = 0.681, p < 0.01, Spearmans rank correlation) and femoral neck (r = 0.632, p < 0.01). No overall loss of bone was shown over the 12 months of follow up, and relative to the reference population there was a significant improvement in BMD at the lumbar spine in women (p < 0.025, paired t test) and at the femoral neck in men (p < 0.05, paired t test). There was a significant negative correlation between the annual percentage change in BMD at the lumbar spine and the duration of gluten free diet (r = -0.429, p<0.01, Spearmans rank correlation), with the largest gain in BMD in patients with most recently diagnosed coeliac disease. Osteoporosis was shown in 47% of patients with treated adult coeliac disease. Recognised risk factors for osteoporosis in the general population including low body mass index, dietary calcium intake, and early menopause are particularly important in coeliac disease. Treatment of coeliac disease with a gluten free diet probably protects against further bone loss, and in the early stages is associated with a gain in bone mineral density.

Expression of inducible nitric oxide synthase activity in human colon epithelial cells: modulation by T lymphocyte derived cytokines

Background—Nitric oxide (NO) synthesis and inducible nitric oxide synthase (iNOS) expression are increased in colonic biopsy specimens from patients with ulcerative colitis, but the cellular source of NO production is not known. Aims—To examine the distribution of iNOS in human colonic mucosa and to explore the ability of T lymphocyte derived cytokines to regulate iNOS expression and activity in human colonic epithelial cells. Methods—iNOS expression was examined using immunohistochemistry in colonic biopsy samples from 12 patients with ulcerative colitis and three with infectious colitis and compared with 10 normal controls. In vitro iNOS expression and activity were determined in HT-29 cell cultures; nitrite levels were measured using a fluorescent substrate, iNOS mRNA expression by northern blot analysis, and iNOS protein expression by western blot analysis. Results—No iNOS expression was detected (10 of 10) in non-inflamed mucosa derived from normal controls. In 11 of 12 cases of newly diagnosed ulcerative colitis, iNOS protein was expressed in the epithelial cells, while no other positive cells were found in the lamina propria. Similar iNOS labelling was found in colonic biopsy samples from patients with infectious colitis in the acute phase, but when re-examined in samples from patients in total remission, no iNOS staining was observed. Both interleukin (IL)-13 and IL-4, but not IL-10, are potent inhibitors of iNOS expression and activity induced by an optimal combination of cytokines, namely IL-1α, tumour necrosis factor α and interferon γ. Conclusions—The data suggest that the epithelium is the major source of iNOS activity in ulcerative colitis and that IL-13 and IL-4 may act as intrinsic regulators of NO generation in intestinal inflammation.

Effect of a gluten free diet on osteopenia in adults with newly diagnosed coeliac disease.

BACKGROUND/AIMS: Calcium and vitamin D malabsorption in coeliac disease predispose to skeletal demineralisation. This study aimed to determine bone mineral density in patients studied in the first year after diagnosis of coeliac disease, and to detect changes in bone mineral density over the subsequent year. METHODS: Lumbar spine and femoral neck bone mineral density was measured in 21 adults with coeliac disease, diagnosed and started on a gluten free diet during the preceding year, with dual energy x ray absorptiometry and repeated after 12 months. RESULTS: Bone mineral density was significantly lower in patients than in paired controls (matched for age and sex), at lumbar spine (0.819 g/cm2 compared with 1.021 g/cm2, p < 0.001 Wilcoxon signed rank test) and femoral neck (0.663 g/cm2 compared with 0.794 g/cm2, p < 0.001). Repeat measurement after 12 months demonstrated that patients had a significant gain in bone mineral density at lumbar spine (16.6%/year), and femoral neck (15.5%/year, p < 0.002, Wilcoxon signed rank test at both sites), whereas no significant change in bone mineral density was detected in controls. CONCLUSIONS: Treatment of coeliac disease with a gluten free diet is associated with a significant increase in bone mineral density, although patients still had lower bone mineral density than controls.

Inducible nitric oxide synthase activity and expression in a human colonic epithelial cell line, HT-29.

1 We have determined which cytokines regulate the expression of human inducible nitric oxide synthase (iNOS) mRNA and nitrite generation in the human colonic epithelial cell line HT‐29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐lα (IL‐lα), tumour necrosisfactor‐α (TNF‐α), interferon γ (IFN‐γ) or vehicle added alone or in combination. Human iNOS mRNA was determined by Northern blot analysis and nitrite generation by the use of a fluorometric assay. 3 Unstimulated cells produced a small time‐dependent increase in nitrite generation of 50 ± 4, 75 ± 8, and 103 ±8 nM per 106 cells at 24 h, 48 h, and 72 h respectively. This nitrite generation was unaffected by cycloheximide (5 μg ml−1) pretreatment and iNOS mRNA was not detected. 4 None of cytokines alone induced either iNOS mRNA expression or an increase in nitrite generation. The combination of IL‐lα/IFN‐γ produced a highly significant (P< 0.001) 4 fold increase in nitrite production at 48 h, compared to basal values, while no other pair of cytokines was effective. 5 Time course studies with IL‐lα/IFN‐γ combination revealed significant (P< 0.001) increases in nitrite at 24 h (153 ±7), 48 h (306 ±24), and 72 h (384 ±15) compared to basal values of 50 ±4, 75 ±8, and 103 ±8 nM per 106 cells respectively. 6 Studies with IL‐lα/IFN‐γ combination demonstrated a time dependent expression of iNOS mRNA, first observed at 6 h, peaked at 24 h and was undetectable by 72 h. IL‐lα (0.3–10 ng ml−1) and IFN‐γ (10–300 u ml−1) in combination induced a concentration‐dependent expression of iNOS mRNA at 24 h. 7 Pretreatment with cycloheximide before IL‐lα/IFN‐γ stimulation reduced nitrite levels to basal values. These data suggest that the IL‐lα/IFN‐γ‐induced nitrite production by HT‐29 cells is dependent on de novo protein synthesis, probably the iNOS enzyme. 8 The addition of TNF‐α produced a significant (P< 0.001) 3 fold increase of IL‐lα/IFN‐γ‐induced nitrite generation. In marked contrast the presence of TNF‐α had no effect on IL‐lα/IFN‐γ‐induced iNOS mRNA expression by HT‐29 cells. These findings suggest that the up‐regulation by TNF‐α of IL‐lα/IFN‐γ‐induced nitrite generation is at the post‐transcriptional level. 9 These data suggest that pro‐inflammatory cytokines induce NO production in colonic epithelial cells probably due to the induction of iNOS and these cells may be a major source of NO generation in inflammatory bowel disease.

C-X-C and C-C chemokine expression and secretion by the human colonic epithelial cell line, HT-29: differential effect of T lymphocyte-derived cytokines

Differential chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected population of leukocytes cells in inflammatory bowel disease. We have previously demonstrated that IL‐13 enhances IL‐1α‐induced IL‐8 secretion by the colonic epithelial cell line HT‐29. We have now explored the C‐C chemokine expression and modulation in this system. The combination of TNF‐α and IFN‐γ was the minimal stimulation required for regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP‐1) mRNA expression and secretion by HT‐29 cells. The same stimulation induced a stronger IL‐8 mRNA expression and secretion. Pretreatment with IL‐13 or IL‐4, reduced significantly the RANTES, and MCP‐1, but not IL‐8 mRNA expression and secretion. In contrast, IL‐10 had no effect on either MCP‐1, or RANTES, or IL‐8 generation. Pretreatment of HT‐29 cells with wortmannin suggested that the IL‐13‐induced inhibition of C‐C chemokine expression is via activation of a wortmannin‐sensitive phosphatidylinositol 3‐kinase. These data demonstrate that colonic epithelial cell chemokine production can be differentially regulated by T cell‐derived cytokines and suggest an interplay between epithelial cells and T lymphocytes potentially important in the intestinal inflammation.

Interleukin-8 production by the human colon epithelial cell line HT-29: modulation by interleukin-13

1 We have determined which cytokines induce and modulate the production of the chemokine interleukin‐8 (IL‐8) by the human colonic epithelial cell line HT‐29. 2 Growth arrested cell cultures were stimulated with the human recombinant cytokines interleukin‐1α (IL‐1α), tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), interleukin‐13 (IL‐13), interleukin‐10 (IL‐10) or vehicle added alone or in combination. The production of IL‐8 was determined by enzyme‐linked immunosorbent assay (ELISA) and IL‐8 messenger RNA expression by Northern blot analysis. 3 The production of IL‐8 in unstimulated cells was undetectable by both ELISA and Northern blot analysis. 4 HT‐29 cells produced IL‐8 following stimulation with IL‐1α or TNF‐α in a time‐ and a concentration‐dependent manner, while IFN‐γ, IL‐10 and IL‐13 did not induce IL‐8 production by HT‐29 cells. 5 IL‐13 was found to up‐regulate significantly (P < 0.01) the IL‐1α but not the TNF‐α‐induced IL‐8 generation by HT‐29 cells. In contrast, IL‐10 had no effect on either IL‐1α or TNF‐α‐induced IL‐8 production. 6 Experiments using cycloheximide demonstrated that this synergistic effect of IL‐13 and IL‐1α on IL‐8 secretion was not through de novo protein synthesis. Using actinomycin‐D, we demonstrated that the IL‐13 up‐regulation was at the level of transcription rather than messenger RNA stability. 7 These findings suggest that colonic epithelial cells have a functional IL‐13 receptor, which is coupled to an up‐regulation of IL‐1α, but not TNF‐α induced IL‐8 generation.

Differential regulation of prostaglandin E biosynthesis by interferon-γ in colonic epithelial cells

Cyclooxygenase (COX)‐2 expression and activity in response to pro‐inflammatory cytokines TNFα and IFNγ was evaluated in the colonic epithelial cell line HT29 and the airway epithelial cell line A549. TNFα induced concentration‐ and time‐dependent upregulation of COX‐2 mRNA, protein and prostaglandin (PG)E2 synthesis. Co‐stimulation of TNFα with IFNγ resulted in reduced COX‐2 mRNA and protein expression. IFNγ had no effect on the stability of TNFα‐induced COX‐2 mRNA. TNFα‐induced PGE2 biosynthesis was significantly enhanced by the simultaneous addition of IFNγ and was COX‐2 dependent. The combination of IFNγ and TNFα induced the microsomal prostaglandin E synthase (mPGES), comensurate with the enhanced PGE2 synthesis. These results suggest that, in terms of PGE2 biosynthesis, IFNγ plays a negative regulatory role at the level of COX‐2 expression and a positive regulatory role at the level of mPGES expression. This may have important implications for the clinical use of IFNγ in inflammatory diseases.

Long term cannabinoid receptor (CB1) blockade in obesity: implications for the development of colorectal cancer.

Dear Sir, An important detail in Greenhough et al. is the response of colorectal cancer cell lines to the CB1 antagonist/inverse agonist, AM251. In this study, the authors provide evidence that the plant-derived cannabinoid, delta(9)-tetrahydrocannabinol (THC), inhibits survival and induces apoptosis in these cells through the CB1 receptor. This endorses the results from Ligresti et al., and Patsos et al., that endogenous and synthetic cannabinoids inhibit colorectal cancer cell growth. The launch of Acomplia (rimonabant) in the UK as an antiobesity treatment has led to much debate regarding the benefits of pharmacotherapy for this condition. Rimonabant is a cannabinoid (CB)-1 receptor antagonist/inverse agonist, which acts both centrally and peripherally to inhibit food intake and regulate metabolic functions at multiple cellular pathways. One major side effect of daily consumption of rimonabant (20 mg) is diarrhea, and one mechanism for this effect is through CB1 antagonism/inverse agonism on enteric nerves, leading to an increase in gut motility. We have reported that the CB1 receptor is normally expressed in the cytoplasm and luminal membrane of human intestinal epithelium, and CB1 agonists have been shown to ameliorate disease severity in animal models of intestinal inflammation. In our studies investigating the mechanism of CB1 and CB2 involvement in human Inflammatory Bowel Diseases (IBD), we have utilised a cell line derived from normal human colonic epithelium, NCM460 and the synthetic CB1 antagonist/inverse agonist, AM251 (Tocris, UK). NCM460 cells have a nontransformed phenotype and may more accurately model the intestinal epithelium than cell lines derived from colorectal tumours. AM251 is structurally and pharmacologically similar to rimonabant and has also been shown to reduce food intake and increase gut motility. In standard proliferation experiments, the daily addition of AM251 had a profound, concentration-dependent antiproliferative effect on NCM460 cells, even at optimal growth conditions in medium complete with serum and growth factors (EC50 5 5.08 lM, Figure 1¤). This effect was more pronounced in low serum conditions (EC50 5 1.84 lM, Figure 1n). Concentrations of AM251 greater than 1 lM have effects that may not be CB1-specific, and the concentration of rimonabant that reaches the intestinal tract in humans is unknown, but this data is in contrast to the published antiproliferative effect of CB1 agonists in colorectal tumour cell lines. Indeed, Greenhough et al., state that AM251 reversed the apoptotic effects of THC in these cells. This highlights the differential effects of cannabinoids on normal versus cancer cell types, but more importantly, we suggest that long term daily consumption of CB1 antagonists during antiobesity treatment could have a negative impact on the integrity and maintenance of the intestinal mucosa, with unknown consequences. Persistent mucosal damage might trigger persistent regenerative responses that predispose susceptible individuals to increased cancer risk, as seen in IBD. Furthermore, the risk of developing colorectal cancer is increased in obesity and sustained CB1 blockade could support the survival of altered intestinal epithelial cells that progress through the adenoma-carcinoma sequence. Equally, it could be argued that unlike bona fide primary intestinal epithelium, NCM460 cells can be cultured in vitro, and thus, could be at an early stage of transformation. Therefore, the point at which CB1 antagonists/inverse agonists switch function is important because they could either prevent the development of aberrant epithelial cells, or promote their survival. Careful analysis and interpretation of CB1-mediated functions, in the FIGURE 1 – Colonic epithelial proliferation in the presence of CB1 antagonist/inverse agonist. About 10 NCM460 cells (¤) were cultured in M3 Base medium (INCELL, San Antonio, TX) with either 10% (n) or 1% (n) serum, and maintained at 37 C/5% CO2 (8). AM251 was added daily at the concentrations indicated. After 96 hr, equating to 3 doubling times, viable cells were counted and numbers expressed as a % of vehicle control cells, n5 6.