Pharmacogenetic biomarkers of response in Crohn's disease.

Crohn's disease (CD) is a chronic condition, which affects the immune system. It can also affect any part of the digestive tract and be associated with external manifestations. The causes of the disease remain unknown, although it seems to be the result of a combination of factors, such as genetic predisposition, environment, lifestyle and the composition of the microbiota, among others. The treatment protocol begins with a change in eating and smoking habits, and is continued with different lines of treatment, including corticosteroids, immunomodulators and biologic therapy (infliximab and adalimumab), which have shown differences in response among patients, especially with biologic treatment. Several studies have considered the possibility that these differences in response are caused by the genetic variability of patients. Many genes have been investigated as potential predictors of response to biological drugs, such as ADAM17, ATG16L1, EMSY, CASP9, CCNY, CNTN5, FASLG, FCGR, NOD2, PTGER4, IL13, IL1B, IL27, IL11, IL17F, TNF and TNFR genes. In this review, we will gather the information on influence of gene polymorphisms investigated to date on response to biological drugs in CD patients.

Nucleotides as semiessential nutritional components.

Dietary nucleotides are required nutrients for some tissues under certain circumstances. A lack of dietary nucleotides negatively influences protein synthesis in both the liver and the small intestine of rats. Ribosome degradation has been observed as being among the mechanisms responsible for this effect. Dietary nucleotides can also modulate gene expression by interaction with specific transcription factors, in both the liver and the small intestine.

Nutritional regulation of nucleoside transporter expression in rat small intestine.

BACKGROUND & AIMS: Concentrative nucleoside transporters CNT1 (pyrimidine preferring) and CNT2 (purine preferring) may be involved in the uptake of nucleoside-derived drugs used in antiviral and chemical therapies. The possibility that nucleoside carrier isoform expression is modulated by nutrient availability has been studied. METHODS: CNT1 and CNT2 tissue distribution was determined by Western blot analysis. The effect of 48-hour starvation on CNT expression was then studied. Nucleoside transporter expression and uptake activity were measured in jejunal brush border plasma membrane vesicles from fed and starved rats. The expression of nucleoside transporters was later determined in a second model of nutrient deficiency: rats fed a purified diet with or without nucleotides for 10 days. RESULTS: CNT1 and CNT2 nucleoside transporters were expressed in a wider variety of tissues than expected from messenger RNA distribution analysis. CNT1 was sensitive to nutrient availability in small intestine and, accordingly, jejunal brush border membrane vesicles from 48-hour-fasted rats showed increased expression of CNT1 and enhanced Na(+)-dependent thymidine and gemcitabine uptake. This effect was mimicked by feeding semipurified diets lacking nucleotides. CONCLUSIONS: Substrate availability modulates nucleoside transporter expression (CNT1) in rat jejunum in vivo.

Hepatotoxic agent thioacetamide induces biochemical and histological alterations in rat small intestine.

We have assessed the effect of the oral ingestion of thioacetamide on small intestine structure and function. Thioacetamide-treated rats showed diminished mucosa weight; protein, DNA, and RNA content; and leucine aminopeptidase activity as compared to controls in both jejunum and ileum. In the jejunum, there was a reduction in the activities of alkaline phosphatase, ATPase, glucose-6-phosphatase, and myeloperoxidase, whereas in the ileum, maltase, lactase, and gamma-glutamyltranspeptidase were reduced. In both jejunum and ileum we found enlarged intercellular spaces, dark epithelial enterocytes, and lymphocyte infiltration. Enterocytes showed lobulated nuclei, deranged mitochondria with loss of their cristae, dilated rough endoplasmic reticulum containing dense material, and vesiculation of the smooth endoplasmic reticulum and the Golgi apparatus. Smooth muscle cells of the intestine exhibited ultrastructural alterations. These findings indicate that chronic oral intake of thioacetamide mimics not only hepatic alterations but also small intestine alterations normally associated with human cirrhosis.

Age-related effect of dietary nucleotides on liver nucleic acid content in rats.

We have addressed the question of whether dietary nucleotides contribute to the liver nucleic acid pool and whether this contribution is related to age. Two experiments were performed in rats of 1 months, 3 months and 17 months of age. In the first, rats were fed a nucleotide-free diet or the same diet, but supplemented with nucleotides for 3 and 10 days. In the second experiment, rats were starved for 3 days. Liver RNA decreased by 10-day deprivation of dietary nucleotides and starvation in young and adult rats but not in the old. Liver DNA decreased by starvation in adult and old rats but not in the young, whereas nucleotide deprivation had no effect. These results, demonstrating that dietary nucleotide deprivation causes an effect on liver RNA pool similar to the effect of starvation, indicate that dietary nucleotides contribute to the liver RNA pool, this influence being related to age.

Morphological changes in hepatocytes of rats deprived of dietary nucleotides.

The aim of the present study was to investigate the influence of dietary nucleotides on liver morphology. Adult rats were fed for 21 d on a nucleotide-containing diet or the same diet free of nucleotides. Liver sections were examined by light and transmission electron microscopy, as well as for nucleic acid and protein contents. Morphometric analysis was performed for different variables. Deprivation of dietary nucleotides resulted in a reduction in hepatocyte nuclear and nucleolar areas as well as in nuclear chromatin condensation. In addition, the rough endoplasmic reticulum was reduced, as were ribosome association and abundance, whereas fat accumulated. These findings portray dietary nucleotides as required nutrients for the liver under normal physiological conditions and suggest that an inadequate supply of nucleotides for a certain period of time has transient negative effects on liver ultrastructure and function.

Deprivation of dietary nucleotides decreases protein synthesis in the liver and small intestine in rats.

BACKGROUND & AIMS: Dietary nucleotides are reported to influence the growth and functioning of the liver and small intestine. The aim of this study was to examine the mechanism by which nucleotides exert their effects in these tissues by assessing protein synthesis activity and related parameters in the presence or absence of dietary nucleotides. METHODS: Rats were fed a purified diet with or without nucleotides for 10 days. Fractional protein synthesis rate, RNA and DNA concentrations, polysome size distribution, and number of ribosomes were assessed. RESULTS: Fractional protein synthesis rates of the liver and small intestine were lower in the nucleotide-deprived group than in the control group. In the liver, RNA concentration was also lower in the nucleotide-deprived group, but values in the small intestine were similar in the two groups. In the liver, deprivation of nucleotides resulted in a reduction in the number of ribosomes and in polysome breakdown. Protein and DNA concentrations did not vary in the liver; however, the concentration of DNA was lower in the small intestine of the nucleotide-deprived group than in the control group. CONCLUSIONS: Dietary nucleotides can modulate protein synthesis in the liver and small intestine as a result of tissue-specific nucleic acid changes.

Changes in plasma lipoproteins and liver lipids in neonatal rats.

Transient increases in triglycerides and cholesterol were found in rat liver immediately after birth. Plasma VLDL and HDL increased after birth and reached a plateau after one week of life. The content of cholesterol ester was low at birth in all lipoproteins and increased in LDL and HDL during the first week of life. After birth, VLDL became enriched in apolipoproteins C and E, whereas HDL was enriched in apolipoprotein C and depressed in apolipoprotein E. The developmental changes in plasma lipoprotein levels and compositions in rats during the first week of life are comparable to those described in humans.

Age-related response of the small intestine to severe starvation and refeeding in rats.

The impact of severe starvation and refeeding on the intestinal mucosa of rats of different ages has been studied in a diet-controlled model. Structural and functional alterations of the small intestinal mucosa were assessed by standard parameters including mucosal protein, DNA content as well as maltase, sucrase and leucine aminopeptidase enzymatic activities. Decreases in mucosal mass, DNA, protein and leucine aminopeptidase activity in both the jejunum and ileum caused by starvation, diminished with age. The depression of disaccharidase activities increased with age in the jejunum but not in the ileum. Except for jejunal protein and leucine aminopeptidase activity, the recovery from starvation, after refeeding, was complete for the other parameters studied, regardless of age.

Deprivation of dietary nucleotides results in a transient decrease in acid-soluble nucleotides and RNA concentration in rat liver.

This study examines the contribution of dietary nucleotides to liver nucleotide pools in rats. Liver acid-soluble nucleotides, DNA and RNA concentrations were monitored in two groups of rats fed either a diet supplemented with nucleotides or a diet free of nucleotides for 3 wk. Significantly lower concentrations of ATP, ADP, GTP and CDP as well as of RNA were found after 1 wk in the rats fed a nucleotide-free diet compared with those fed the nucleotide-supplemented diet; concentrations remained lower after 2 wk except for ATP and ADP. No changes over time were observed in the rats fed the nucleotide-supplemented diet. Between wk 2 and 3 an increase in both acid-soluble nucleotides and RNA was observed in the rats fed the nucleotide-free diet, reaching the values found in the rats fed the nucleotide-supplemented diet. These findings, which indicate that dietary nucleotides are utilized at least in part by the liver to maintain the cell nucleotide pools and that diets devoid of nucleotides affect hepatic nucleotide metabolism and RNA, support the hypothesis that liver nucleotide metabolism is modulated by the availability of dietary nucleotides.

Dietary nucleotides accelerate intestinal recovery after food deprivation in old rats.

Previous studies in very young rats have shown that dietary nucleotides improve small intestine repair after injury or malnutrition. To investigate the potential effect of nucleotides in old rats, which have a diminished capability for intestinal repair, 17-mo-old rats were deprived of food for 5 d and then fed a nucleotide-free diet or a nucleotide-supplemented diet for 3 or 6 d. Intestinal jejunal and ileal mucosal weight, protein and DNA were evaluated as intestinal growth markers, and brush-border maltase, sucrase, lactase and aminopeptidase activities were evaluated as intestinal differentiation markers. The adenine nucleotide pool and the adenylate energy charge were also evaluated as indices of nucleotide availability. Food deprivation significantly decreased mucosal growth markers as well as differentiation markers in both jejunum and ileum. The ATP pool was also significantly depressed, but the adenylate energy charge was not significantly altered. To a certain extent, refeeding restored the losses, but in the rats that were fed the nucleotide-free diet, the restoration of the jejunum was significantly slower and the restoration of the ileum differentiation markers was incomplete compared with the rats fed the nucleotide-supplemented diet. The results suggest that dietary nucleotide intake in the elderly may accelerate the normal physiological intestinal response to refeeding after food deprivation.

Dietary nucleotides enhance plasma lecithin cholesterol acyl transferase activity and apolipoprotein A-IV concentration in preterm newborn infants.

The activity of lecithin cholesterol acyl transferase (LCAT), a key enzyme in lipoprotein metabolism, is low in newborn preterm infants. It has been suggested that a normal gastrointestinal function might be necessary to induce a postnatal increase of LCAT activity because apoproteins A-I and A-IV (apoA-I and apoA-IV) synthesized in considerable amounts in the intestine are known activators of LCAT. Dietary nucleotides have been reported to enhance intestinal growth and maturation; therefore, we hypothesized that nucleotide supplementation to formulas for preterm infants may influence LCAT activity. To investigate this hypothesis, two groups of preterm infants were fed either a nucleotide-free formula or a nucleotide-supplemented formula during the first month of life. The plasma LCAT activity, plasma levels of apoA-I and apoA-IV, plasma cholesteryl esters, and plasma fatty acid composition of cholesteryl esters and phospholipids were then determined. Infants receiving nucleotides had higher LCAT activities and apoA-IV levels than those receiving the nucleotide-free formula for a few weeks. The changes in apoA-IV levels were highly correlated with those of the LCAT activities. However, there were no significant correlations between changes in LCAT activity and plasma cholesteryl esters or phospholipids. These findings indicate that nucleotide supplementation to formulas for preterm infants may improve dietary lipid tolerance by enhancing plasma LCAT activity, probably as a result of an increase in apoA-IV plasma concentrations; they also suggest that nucleotides may enhance apoA-IV synthesis in the intestine during the neonatal period.

Maturation status of small intestine epithelium in rats deprived of dietary nucleotides.

We describe the changes of several brush-border enzymatic activities in different subpopulations of epithelial cells, separated sequentially from the villus tip-to-crypt axis of the small intestine, induced by deprivation of dietary nucleotides for different periods of time in adult rats. Deprivation of dietary nucleotides lead to a decrease in the content and specific activity of alkaline phosphatase, leucine-aminopeptidase, maltase, sucrase and lactase in the villus tip, but had little effect on the crypt zone. The effect of the nucleotide deprivation on the enzymatic activity progressively increased towards the tip of the villus. Since these enzymes are maturation markers of the intestinal cells, these results support the idea that dietary nucleotides affect the maturation status of small-intestine epithelium.

Lipoprotein changes in small-for-gestational-age infants fed nucleotide-supplemented milk formula.

We determined the effect of supplementing milk formula with nucleotides on plasma lipoproteins in small-for-gestational-age infants: 21 infants were fed a nucleotide-supplemented formula and 20 infants were fed the same nucleotide-free formula. On days 0, 3 and 7 after birth, major plasma lipoprotein fractions were analyzed for apolipoprotein and lipid composition. Compared with the control group, the group receiving nucleotides had increased total apoprotein concentrations in all lipoproteins as well as increased apo A-I in high-density lipoproteins and very low-density lipoproteins, and apo B-100 in very low-density lipoproteins and low-density lipoproteins. Very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol and very low-density lipoprotein triglycerides increased in parallel to the changes in apoproteins. The cholesterol ester to unesterified cholesterol ratio was increased in low-density lipoproteins and, particularly, in high-density lipoproteins. These data support the hypothesis that lipoprotein metabolism in small-for-gestational-age infants is affected by dietary nucleotide supplementation, enhancing lipoprotein synthesis or secretion. Cholesterol esterification capacity paralleled the apo A-I increase, in agreement with the cofactor role of apo A-I on lecithin: cholesterol acyltransferase.

Dietary nucleotides influence lipoprotein metabolism in newborn infants.

Nucleotide supplementation of adapted-milk formulas may be of interest for infant nutrition because nucleotides are involved in the synthesis of proteins and other macromolecules such as phospholipids, and thereby facilitate lipoprotein synthesis. To determine whether dietary nucleotides influence plasma lipoproteins in newborns, we have studied the plasma-lipoprotein concentrations and the composition of the major lipoprotein fractions during the first week of life in two groups of preterm infants fed formulas differing only in their nucleotide content. For comparison, two groups of term infants were studied under the same conditions. Lipoproteins were isolated by density ultracentrifugation, and the lipid and protein content were determined by standard methods; apolipoprotein A-I was determined immunologically. Nucleotide supplementation of formula in preterm infants increased all plasma lipoprotein concentrations. In addition, an increase in the plasma esterification rate was observed. However, total cholesterol concentrations were unchanged. The changes in lipoproteins concentrations were due mainly to an increase in apolipoprotein content. Nucleotides added to formulas affected term-infants' lipoproteins significantly less than to preterm infants. These findings suggest that dietary nucleotides may enhance the synthesis of lipoproteins during the early neonatal period, especially in preterm infants.

Lipoproteins in preterm and small-for-gestational-age infants during the first week of life.

Plasma lipoprotein levels and composition have been determined in preterm and small-for-gestational-age (SGA) infants, and compared to full-term infants, during the first week of life. Significantly lower levels of HDL and higher levels of VLDL were found in both preterm and SGA infants in comparison to full-term healthy infants. These results suggest a low capacity to metabolize VLDL. Preterm infants showed a behaviour similar to full-term infants with regard to the changes in lipoprotein composition. Small-for-gestational-age infants showed a higher lipoprotein lipid content than preterm infants. A low ratio of cholesteryl ester to free cholesterol (CE/FC) was found in both preterm and SGA infants suggesting a reduced lecithin: cholesterol acyl transferase (LCAT) activity. In preterm infants we observed no changes in the CE/FC ratio during the first week of life, whereas in SGA infants this ratio increased after birth.

Antihypercholesterolemic effect of dipyridamole in chickens.

The administration of a 4 mg/kg dose of dipyridamole daily in chickens fed a diet supplemented with 2% cholesterol reversed the hypercholesterolemic effects of the diet. In particular, it reduced the plasma cholesterol concentration in approximately 18%; the levels of very-low-density lipoproteins and intermediate-density lipoproteins and the liver cholesterol content. Although the mechanism was not fully elucidated, the increased excretion of cholesterol seemed to be responsible for the lipid lowering effect. When dipyridamole was administered in chickens fed the same diet without cholesterol no significant changes were observed. Inasmuch as the chicken lipoprotein metabolism differs in several aspects to human, the extrapolation of the hypocholesterolemic effect of dipyridamole to man must be made with care.

A regulatory element is characterized by purine-mediated and cell-type-specific gene transcription.

Purines and purine nucleotides were found to affect transcription of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in whole nuclei isolated from intestinal mucosa of adult rats fed a purine- and purine nucleotide-free diet. Nuclear run-on transcription assays, performed on whole nuclei from different tissues and cell types, identified an intestine-specific decrease in the overall incorporation of [alpha-32P]UTP in HPRT transcripts from intestinal epithelial cell nuclei when exogenous purines or purine nucleotides were omitted from either the diet or culture medium. Using a 990-base-pair genomic fragment that contains the 5'-flanking region from the HPRT gene, we generated plasmid constructs with deletions, transfected the DNA into various cell types, and assayed for chloramphenicol acetyltransferase (CAT) reporter activity in vitro. We determined that an element upstream from the putative transcriptional start site is necessary to maintain the regulatory response to purine and nucleotide levels in cultured intestinal epithelial cells. These results were tissue and cell type specific and suggest that in the absence of exogenous purines, the presence of specific factors influences transcriptional initiation of HPRT. This information provides evidence for a mechanism by which the intestinal epithelium, which has been reported to lack constitutive levels of de novo purine nucleotide biosynthetic activity, could maintain and regulate the salvage of purines and nucleotides necessary for its high rate of cell and protein turnover during fluctuating nutritional and physiological conditions. Furthermore, this information may provide more insight into regulation of the broad class of genes recognized by their lack of TATA and CCAAT box consensus sequences within the region proximal to the promoter.

Metabolic changes induced by urethane-anesthesia in rats.

1. Fasting hyperglycemia was observed in urethane-anesthetized rats. No significant changes had been observed in fed animals. The effect is dose-dependent, being ineffective doses lesser than 1.4 g/kg of body weight. 2. Urethane originates a rise in glycemia during the first 10 min of anesthesia followed by control values at 30 min, and a latter hyperglycemic phase for more than 60 min that remain at 2 hr. 3. The negative correlationship between plasma glucose, lactate and amino acid levels suggest that gluconeogenesis may be the main responsibility of the observed hyperglycemia during the first phase, but it is possible that during the second phase a decrease in the consumption of glucose may take place as a consequence of the competitive effects of ketone bodies increased during the first 30 min of anesthesia. 4. We postulate that the mechanism of the hyperglycemic response to urethane is a sympathetic response with release of catecholamines both in the liver and in the adrenal gland which enhances gluconeogenesis and lipolysis.