Factors associated with nonadherence to thiopurines in adolescent and adult patients with inflammatory bowel disease.

OBJECTIVES: We hypothesised that nonadherence to thiopurines is more common in adolescents than in adults with inflammatory bowel disease. METHODS: We sought factors associated with thiopurine nonadherence defined by thiopurine metabolite levels. RESULTS: Multivariate logistic regression confirmed that adolescents (odds ratio [OR] 4.6 [95% confidence interval [CI] 1.9-11.5]; P < 0.01) compared with adults, patients with Crohn disease (OR 3.3 [CI 1.1-10.5] P = 0.04) compared with ulcerative colitis, and patients living in more socially deprived areas (OR 1.03 [CI 1.0-1.1] P = 0.02) were more likely to be nonadherent to thiopurines. CONCLUSIONS: Adolescents are more frequently nonadherent than adults: prospective studies are required to determine the reasons for nonadherence in adolescents.

Do children with IBD really respond better than adults to thiopurines?

BACKGROUND AND OBJECTIVES: Children and adolescents with inflammatory bowel disease (IBD) have more extensive and severe disease than adults. Despite a lack of comparative studies, thiopurines are frequently cited as being more efficacious in children. To test this assertion, we compared the efficacy of thiopurines in children with IBD with that in adults matched for disease phenotype. PATIENTS AND METHODS: Fifty paediatric and adult patients with IBD started on a thiopurine were matched for sex, disease type, and extent. Retrospective data were obtained by electronic case note review, and corticosteroid-free clinical remission and tolerance rates at 6 months as well as relapse rates during the subsequent year were recorded. RESULTS: Adverse effects caused discontinuation of thiopurines in 1 of 50 children and 16% (8/50) of adults (P < 0.05). At 6 months, steroid-free remission was achieved in 30% (15/50) of children and 38% (19/50) of adults (P = 0.53). No differences in remission rates were seen according to disease type. At the end of the following year, 73% (11/15) of children and 68% (13/19) of adults remained in remission (P = 1). CONCLUSIONS: Thiopurines are tolerated better by children. When phenotype is matched, there is no difference in the therapeutic response to thiopurines between children and adults with IBD.

Metabolic studies in total parenteral nutrition with lipid in man. Comparison with glucose.

A study was undertaken of patients on a regimen of total parenteral nutrition comparing the nitrogen balance, energy substrates, blood amino acids, immunoreactive insulin, and immunoreactive glucagon levels during the sequential infusion of nonprotein calories as either glucose alone (glucose system) or 83% as Intralipid (Pharmacia Fine Chemicals, Montreal, Canada) and 17% glucose (lipid system). These nonprotein calories were administered with a constant background of amino acids (1 g/kg per day), vitamins, and minerals. Each system was infused for a week at a time and the order of infusion randomized. In some patients whole blood arteriovenous (A-V) levels of amino acids were measured across forearm muscle. During the glucose system there was a significantly higher level of pyruvate, lactate, alanine, and immunoreactive insulin, consistent with glucose being the principal source of energy. In contrast, during the lipid system there was a rise in free fatty acids and ketone bodies with a fall in insulin, suggesting that lipid was now the principal source of energy. Despite these two very diverse metabolic situations the nitrogen balance with both systems was positive to a comparable degree after the establishment of equilibrium. Correspondingly, A-V differences of whole blood amino acid nitrogen showed uptake by muscle to an equivalent degree with both systems. Clinical studies indicated that the lipid system as defined herein could be infused by peripheral vein for up to 43 days with resultant weight gain, elevation of serum proteins, and healing of fistulae. Our studies suggest that for both metabolic and clinical reasons exogenously infused lipid is a suitable source of nonprotein calories.

Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability.

Enzymes from hyperthermophilic organisms must operate at temperatures which rapidly denature proteins from mesophiles. The structural basis of this thermostability is still poorly understood. Towards a further understanding of hyperthermostability, we have determined the crystal structure of the beta-glycosidase (clan GH-1A, family 1) from the hyperthermophilic archaeon Sulfolobus solfataricus at 2.6 A resolution. The enzyme is a tetramer with subunit molecular mass at 60 kDa, and crystallises with half of the tetramer in the asymmetric unit. The structure is a (betaalpha)8 barrel, but with substantial elaborations between the beta-strands and alpha-helices in each repeat. The active site occurs at the centre of the top face of the barrel and is connected to the surface by a radial channel which becomes a blind-ended tunnel in the tetramer, and probably acts as the binding site for extended oligosaccharide substrates. Analysis of the structure reveals two features which differ significantly from mesophile proteins; (1) an unusually large proportion of surface ion-pairs involved in networks that cross-link sequentially separate structures on the protein surface, and (2) an unusually large number of solvent molecules buried in hydrophilic cavities between sequentially separate structures in the protein core. These factors suggest a model for hyperthermostability via resilience rather than rigidity.

Ontogeny of class II MHC mRNA in the mouse small intestinal epithelium.

MHC Class II (Ia) and invariant chain cooperate in the presentation of exogenous antigen by antigen presenting cells to T-helper cells. Both glycoproteins have been identified in the small intestine of the mature mouse. In this study, we examine the ontogeny of mRNA for three molecules; (Ii31, Ii41 and I-A beta) in whole intestine and in isolated epithelial cells. When RNA from whole intestine was analysed in northern blots using cDNA probe, Ii31 mRNA was present in Day 10 mice and at each 5 day time point thereafter; Ii41 and I-A beta were not detected by this technique. To examine ontogeny of Ii chain mRNA in enterocytes, RNA was purified from an enriched population of epithelial cells isolated after systemic perfusion with 30 mM EDTA in Day 21 and Day 28 and adult mice. Ii chain mRNA was not detected until Day 28 by blot hybridization. Reverse transcription of mRNA and amplification of the resultant cDNA by PCR revealed Ii41 and I-A beta as well as Ii31. RNA from Day 21 epithelial cells required five additional amplification cycles to attain cDNA levels equivalent to those found in Day 28 cells for Ii chain, and 10 additional cycles for I-A beta. In conclusion, Ii31, Ii41 and I-A beta mRNA increase rapidly in the enterocyte after weaning.

Dietary regulation of genes expressed in the developing intestinal epithelium.

Gene expression is central to the pathogenesis of many disorders. An ability to alter the expression of genes would, if their relation to disease processes were fully understood, constitute a new modality of treatment. This lecture examines the evidence that nutritional factors can regulate genes in the gastrointestinal epithelium and it discusses the physiologic relevance of such alterations in gene expression. Dietary regulation of the genes expressed by the epithelium confers 3 fundamental advantages for mammals. It enables the epithelium to adapt to the luminal environment to better digest and absorb food; it provides the means whereby breast milk can influence the development of the gastrointestinal tract; and, when the proteins expressed by the epithelium act on the immune system, it constitutes a signaling mechanism from the intestinal lumen to the body's defenses. Each of these mechanisms is amenable to manipulation for therapeutic purposes.

The physicochemical environment of the neonatal intestine.

Dietary intake, bacterial metabolites, and the secretion of factors (eg, proteins, electrolytes, lipid-soluble molecules, and water) by the body each contribute to the physicochemical environment of the gastrointestinal tract. Peristalsis regulates the changes along the length of the intestine. However, coordinated peristaltic responses develop as premature infants mature. In addition, the physicochemical environment of the center of the intestinal lumen differs from that of the epithelial surface. The area adjacent to the small intestinal epithelium is more acid than the bulk phase. Na+/H+ exchange antiporters in the epithelial cell apical membrane generate this acidity. Mucus maintains the acid microclimate by preventing free diffusion of hydrogen ions into the bulk phase. Development also affects these mechanisms. Changes in the lumenal environment may alter the synthesis of signaling molecules expressed by the intestinal epithelium. Thus, the epithelium, through changes in gene regulation, may act as an active interface that transmits information about the composition of the intestinal lumen to the mucosal immune system. Premature neonates are at risk of necrotizing enterocolitis, a disease almost exclusively associated with oral feeds. The pathogenesis of this condition may, in part, be due to the immaturity of the interactions between the physicochemical environment of the lumen and intestine.

3-Acyl-4-hydroxyquinolin-2(1H)-ones. Systemically active anticonvulsants acting by antagonism at the glycine site of the N-methyl-D-aspartate receptor complex.

Most full antagonists at the glycine site of the NMDA receptor contain a carboxylic acid, which we believe to be detrimental to penetration of the blood-brain barrier. By consideration of a pharmacophore, novel antagonists at this site have been designed in which the anionic functionality is a vinylogous acid, in the form of a 4-hydroxyquinolin-2(1H)-one. In this series, a 3-substituent is necessary for binding, and correct manipulation of this group leads to compounds such as the 3-(3-hydroxyphenyl)propargyl ester 24 (L-701,273), with an IC50 for displacement of [3H]-L-689,560 binding of 0.17 microM and Kb against NMDA in the cortical slice of 1.39 microM. Compounds were tested for their ability to prevent audiogenic seizure in DBA/2 mice; the most potent compound in this series is the cyclopropyl ketone 42 (L-701,252), with an ED50 of 4.1 mg/kg ip. A model is proposed for binding to the glycine site, in which an important interaction is of a putative receptor cation with the pi-system of the 3-substituent.

Tryptophan-derived NK1 antagonists: conformationally constrained heterocyclic bioisosteres of the ester linkage.

The 3,5-bis(trifluoromethyl)benzyl ester of N-acetyl-L-tryptophan 1 (L-732,138) has been identified previously as a potent and selective substance P receptor antagonist. A series of analogs which introduced a 6-membered heterocyclic ring into the backbone of this structure were prepared for evaluation as bioisosteric replacements of the ester linkage of 1. The 2,5-dioxopiperazine 2 had very weak receptor affinity, but 2-oxopiperazine 5 exhibited modest activity. Examination of the conformations accessible to the substituents on these templates led to exploration of the corresponding 5-membered heterocyclic rings. This study culminated in the identification of oxazolidinedione 14 as a suitable ester mimic in terms of the retention of good NK1 binding affinity.

Interleukin-1beta (IL-1beta) and IL-6 modulate insulin-like growth factor-binding protein (IGFBP) secretion in colon cancer epithelial (Caco-2) cells.

Chronic inflammation is characterised by modifications in cytokine concentrations, whereas growth is mainly dependent on the GH-IGF axis. IGF-I bioavailability is modulated by a family of IGF-binding proteins (IGFBPs). The aim of the present study was to evaluate the interactions among interleukin-1beta (IL-1beta), IL-6 and IGFBP secretion by intestinal cells to assess whether cytokines modulate IGFBP secretion, and in turn IGF-I and IGF-II bioavailability. The human colon carcinoma derived cell line Caco-2 was used as an in vitro model for its capacity to differentiate spontaneously. Experiments were carried out on day 4 (undifferentiated state) and day 14 (differentiated state) after plating. Carcinoembryonic antigen (CEA) was used as a marker of differentiation and increased in the conditioned media (CM) from days 4 to 14 (0.2+/-0.01 ng/ml per 10(5) cells vs 3.3+/-0.2 ng/ml per 10(5) cells, P<0.05). IGFBP-2 and IGFBP-4 secretion decreased concomitantly. Cells were stimulated with IL-1beta and IL-6 at 1, 10 and 50 ng/ml, and with IL-1beta and IL-6 in combination at the same dose of 1 and 10 ng/ml. IGF-I at 50 ng/ml was used as a control. Caco-2 cells expressed and secreted mainly IGFBP-2 and IGFBP-4 into the CM. On day 4, IL-1beta (1 ng/ml) and IL-6 (10 and 50 ng/ml) reduced IGFBP-2 by 29+/-8%, and by 32+/-9 and 38+/-8% respectively (P<0.05). IGFBP-4 was also reduced by IL-1beta at 1 and 50 ng/ml (-14+/-4% and -46+/-11% vs serum free medium (SFM) respectively, P<0.05), and IL-6 at 50 ng/ml (-46+/-15%, P<0.05). Both IGFBP-2 and IGFBP-4 were reduced by IL-1beta and IL-6 in combination at 1 and 10 ng/ml (P<0.05). On day 14, IGFBP-2 band intensity was reduced at 10 ng/ml of IL-1beta (-22+/-15% vs SFM, P<0.05) and at 50 ng/ml of both cytokines (-33%+/-8% and -13%+/-13% vs baseline respectively, P<0.05). IGFBP-4 band intensity decreased with 10 and 50 ng/ml of IL-1beta (-35+/-11% and -46+/-15% vs SFM respectively) and IL-6 (-36%+/-10% and -46+/-15% vs SFM respectively). IL-1beta and IL-6 in combination at 1 and 10 ng/ml reduced both IGFBP-2 and IGFBP-4.In conclusion, IGFBP-2 and IGFBP-4 secretion in CM decreased with Caco-2 cell differentiation. IGFBP-2 and IGFBP-4 were significantly decreased by IL-1beta and IL-6 treatment in both the undifferentiated and differentiated state. Furthermore, these cytokines increased cell proliferation whereas total protein content was significantly reduced only at the higher concentrations of IL-6 and IL-1beta. These findings suggest that interleukins modulate the IGF-IGFBP system in Caco-2 cells in vitro.

The central role of chemokines (chemotactic cytokines) in the immunopathogenesis of ulcerative colitis and Crohn's disease.

The final composition of leukocytes present in a site of inflammation in response to chemokine stimulation and activation may depend on both the nature of the secreted chemokines as well as the relative expression of the multitude of specific chemokine cell surface receptors on many different cell types. Because related receptors with different affinities and cross-reactive binding capabilities are present on each type of leukocyte, relative differences in receptor distribution and receptor affinity for specific chemokines may significantly influence which cells are ultimately attracted to and activated by each individual chemokine. Production of IL-8, MCP-1, and ENA-78 by endothelial cells, LPMNC, and epithelial cells in IBD could establish a chemotactic gradient capable of influencing the increased migration of monocytes/macrophages, granulocytes, and lymphocytes from the blood stream through the endothelium into both the mucosa and submucosa during chronic IBD. The ability of chemokines to induce chemotaxis, leukocyte activation, granule exocytosis, increased production of metalloenzymes, and up-regulation of respiratory burst activity indicates that there may be a variety of different mechanisms by which chemokines could markedly increase chronic inflammation and chronic intestinal tissue destruction in IBD.

Density of mucosal mast cells in the lamina propria of the colon and terminal ileum of children.

Ileal and colonic biopsies from children observed to be histologically normal were fixed in Carnoy's fluid, sectioned, and stained by the chloroacetate esterase reaction. The density of mucosal mast cells was higher in the terminal ileum than the colon, but did not vary significantly within the colon.

Lamina propria mast cells in biopsies from children with Crohn's disease.

Biopsies from actively inflamed areas of terminal ileum or colon in children with Crohn's disease were examined both for lamina propria mast cell density and histamine content. These were reduced in comparison with those of normal controls. The release of histamine from biopsies of inflamed tissue did not differ greatly from that of normal tissue, either spontaneously or after receiving an antihuman IgE challenge.

Chemotherapy as the initial treatment of spinal cord compression due to disseminated neuroblastoma.

During a 12-month trial period, all children attending the Hospitals for Sick Children, London, England, for management of spinal cord compression due to disseminated neuroblastoma were given chemotherapy as initial treatment rather than radiotherapy or laminectomy. Response to treatment was evaluated by a neurosurgeon as well as by oncologists. Four children were treated in this way and all made a full recovery of spinal cord function.

The handling of glucose and insulin response before and after weight loss with jejuno-ileal bypass: a preliminary report.

Glucose and insulin response were studied in morbidly obese patients before and after jejuno-ileal bypass. Postbypass studies were performed after weight loss greater than 30% (9-48 mon). Six-hour oral glucose tolerance tests were performed on 33 patients before bypass and on 13 of these 33 patients after bypass. Thirteen patients had preoperative intravenous glucose tolerance tests performed, and 11 had postbypass intravenous glucose tolerance tests performed. Plasma insulins were drawn concomitantly. Before bypass, fasting blood sugars and insulins were elevated and both glucose intolerance and hyperinsulinemia were found on the tests. After bypass, oral glucose tolerance test curves were flat (malabsorption effect), while intravenous glucose tolerance tests revealed nonsignificant changes in the glucose curves but plasma insulin response returned toward normal (p greater than 0.05). The glucose intolerance and hyperinsulinemia found in these massively obese patients reverted toward normal after weight loss from jejuno-ileal bypass. Thus elevated plasma insulin does not appear to be a primary etiological factor in obesity.

Development of immune function in the intestine and its role in neonatal diseases.

This review has traced the ontogeny of the human mucosal immune system, speculating that appropriate gut immune responses are essential in preventing many significant neonatal enteric diseases. Because the gastrointestinal tract serves as the portal of entry for many potential antigens, its mucosal immune function is essential in controlling antigenic responses and ensuring systemic tolerance. A thorough under standing of the development of the entire immune system is essential in defining intestinal mucosal immune function. From the protective barrier covering the enterocyte to the intraepithelial T lymphocytes, these components work together to limit antigen passage from the gut lumen to the underlying immune cells and, thus, promote normal immunity and tolerance. When abnormalities exist or when this immune barrier has not matured fully, conditions afflicting newborns, especially preterm infants, occur. Necrotizing enterocolitis, milk-protein enteropathy, and enteric bacterial infections are only three clinical examples of how aberrant gut immune-mediated defenses may have a significant role in their pathogenesis. In clinical practice, it is not only important to recognize these conditions at their onset but also to understand the basis for the underlying illness and identify newborns who are at an increased risk of acquiring them.

An evaluation of the relative importance of formulation and process variables using factorial design.

A factorial design method for assessing the relative importance of various formulation and process factors and their interactions in model paracetamol tablets is described. The design was a 2 X 2 X 2 X 3 type using mixing time, starch concentration, drug particle size and compaction pressure respectively. The starch concentration was the most significant factor in affecting the dissolution rate but the larger drug particle size also gave a significant increase in drug release rate. Interactions between starch concentration and drug size and between these and mixing time were also observed. The most significant factor affecting the tensile fracture stress of the tablets was the mixing time, followed in order by the drug particle size, starch concentration and compaction pressure.

Growth in Crohn's disease.

Abnormal linear growth is frequent in children and adolescents with Crohn's disease. The typical pattern is of growth retardation associated with delayed skeletal maturation. Puberty is also frequently delayed. Over 50% of patients may have a subnormal height velocity, and approximately 25% will have short stature. The endocrine status is characterized by normal growth hormone secretion and a slightly subnormal serum level of insulin-like growth factor I, which is related to nutritional status. Principal therapeutic options are intestinal resection for localized disease, and enteral nutrition--using a polymeric diet--for more widespread disease, particularly involving the small intestine. Growth responses to both modalities are often excellent and produce considerable psychological benefit. Optimum therapy is achieved by close collaboration between gastroenterologists and endocrinologists, and by the use of auxological methods to document pre- and post-therapeutic management.

Constitutive TLR4 signalling in intestinal epithelium reduces tumor load by increasing apoptosis in APC(Min/+) mice.

The microbial pattern-recognizing Toll-like receptors (TLRs) are major signal transducers known to shape and influence the postnatal maturation of host intestinal epithelium. Perturbations in this intricate host-microbe cross-talk have been reported to be associated with uncontrolled epithelial cell growth and thus potential cancer development by mechanisms which are largely unknown. We therefore generated transgenic mice carrying a constitutively active TLR4 (CD4-TLR4) linked to an intestinal epithelial cell-specific promoter. Ex vivo analysis of transgenic crypt-villus organoid cultures revealed an increased proliferative capacity and a lowered cyclooxygenase 2 (Cox-2) expression in these organoids compared with wild-type control cultures. Introducing the CD4-TLR4 transgene into APC(Min/+) mice (CD4-TLR4-APC(Min/+)), a model of colorectal carcinoma, resulted in a dramatic drop in tumor load as compared with control APC(Min/+) mice. Intestinal tumors from CD4-TLR4-APC(Min/+) mice displayed reduced Cox-2 protein, elevated interferon ß expression and increased caspase-3 activity, which correlated with increased apoptosis in vivo. Thus, our data reveal that host microbiota-mediated signal transduction via TLR4 in intestinal epithelial cells is far more complex than what is previously reported.

Mathematical modelling to restore circulating IGF-1 concentrations in children with Crohn's disease-induced growth failure: a pharmacokinetic study.

OBJECTIVES: Children with Crohn's disease grow poorly, and inflammation depresses the response of insulin-like growth factor-1 (IGF-1) to growth hormone. Correcting the inflammation normalises growth velocity; however, removing inflammation cannot be achieved in all children. Our lack of understanding of IGF-1 kinetics has hampered its use, particularly as high IGF-1 concentrations over long periods may predispose to colon cancer. We hypothesised that mathematical modelling of IGF-1 would define dosing regimes that return IGF-1 concentrations into the normal range, without reaching values that risk cancer. DESIGN: Pharmacokinetic intervention study. SETTING: Tertiary paediatric gastroenterology unit. PARTICIPANTS: 8 children (M:F; 4:4) entered the study. All completed: 5 South Asian British; 2 White British; 1 African British. INCLUSION CRITERIA: Children over 10 years with active Crohn's disease (C reactive protein >10 mg/l or erythrocyte sedimentation rate >25 mm/h) and height velocity <-2 SD score. EXCLUSION CRITERIA: closed epiphyses; corticosteroids within 3 months; neoplasia or known hypersensitivity to recombinant human IGF-1 (rhIGF-1). INTERVENTIONS: Subcutaneous rhIGF-1 (120 µg/kg) per dose over two admissions: the first as a single dose and the second as twice daily doses over 5 days. PRIMARY OUTCOME: Significant increase in circulating IGF-1. SECONDARY OUTCOMES: Incidence of side effects of IGF-1. A mathematical model of circulating IGF-1 (Ac) was developed to include parameters of endogenous synthesis (Ksyn); exogenous uptake (Ka) from the subcutaneous dose (As): and IGF-1 clearance: where dAc/dt=Ksyn - Kout×Ac+Ka×As. RESULTS: Subcutaneous IGF-1 increased concentrations, which were maintained on twice daily doses. In covariate analysis, disease activity reduced Ksyn (p<0.001). Optimal dosing was derived from least squares regression fitted to a dataset of 384 Crohn's patients, with model parameters assigned by simulation. CONCLUSIONS: By using age, weight and disease activity scaling in IGF-1 dosing, over 95% of children will have normalised IGF-1 concentrations below +2.5 SDs of the normal population mean, a level not associated with cancer risk.