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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Protein composition of human milk in relation to mothers' weight and socioeconomic status.

In the past few years there has been a resurgence of interest in the protein composition of human milk. Up to now the influence of maternal diet and of the mothers' nutritional status on the protein composition of human milk have not been fully clarified. We have evaluated the relationship between the mothers' socioeconomic status and weight and the protein composition of human milk. Protein fractions were determined by a polyacrylamide gel electrophoresis method in 181 samples of human milk obtained from voluntary donors. Samples were classified according to the time of lactation and in relation to the socioeconomic status and to the weight of the lactating women. Total protein and non-protein nitrogen decreased with advancing lactation but there were no differences among the socioeconomic and weight groups of mothers who were considered. beta- and kappa-caseins fell during lactation and beta-casein was significantly increased in the milk of the upper socioeconomic class with respect to that of the low one. alpha-lactalbumin increased from transitional to mature milk (16-30 d) and then declined. The milk from the low socioeconomic group presented the lowest levels of this protein. Lysozyme increased during lactation, whereas lactoferrin decreased. Both proteins were significantly influenced by the mothers' socioeconomic status; the highest concentrations for these proteins were found in the milk of the low socioeconomic group. Deficit or excess of mothers' weight did not influence the levels of the different protein fractions of human milk.

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.

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.

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.

Role of renal gluconeogenesis in the maintenance of glycaemia after L-tryptophan administration to rats.

The time-course of liver and kidney gluconeogenesis after L-tryptophan administration has been studied. Two and half hours after injection of L-tryptophan (0.5 g/kg body wt) a 97% inhibition of hepatic gluconeogenesis in starved rats was observed. Twelve hours later, the inhibition remained 35%. Hepatic glycogen was almost completely depleted (97%) in fed rats after 5 hours. At this time there was a severe hypoglycaemia in fed and 48 h starved rats which gradually disappeared with time, the values going back to normal after 12 hours. Tryptophan treatment was associated with a significant increase in renal gluconeogenesis in fed and 48 h starved rats with a maximum at 5 h (165% and 190% respectively). When hepatic gluconeogenesis was constantly inhibited in fed rats by periodic injection (every 4 h) of L-tryptophan, renal gluconeogenic ability remained increased throughout the experiment while blood glucose concentrations did not change. These observations suggest that kidney contributes to maintain glycaemic homeostasis under these conditions of liver gluconeogenesis impairment.

[Selective inhibition of hepatic gluconeogenesis gy tryptophan administration]. / Inhibición selectiva de la gluconeogénesis hepática por administración de triptófano.

The levels of quinolinic acid in liver and kidney and the gluconeogenic capacity in these tissues were determined in rats treated with tryptophan. The administration of this aminoacid produced a high increase in the hepatic quinolinic acid concentrations fed and 48 h. starved rats. On the contrary, only slight variations in the concentrations of renal quinolinic acid were observed. The highest value raised in these conditions was three times lower than Ki of the quinolinic acid for the phosphoenolpyruvate carboxykinase. These results suggest that tryptophan administration originates a selective inhibition of hepatic gluconeogenesis.

[Evolution of the content of gluconeogenic metabolites in rat liver and kidney during exercise (author's transl)]. / Evolución de la concentración de los metabolitos gluconeogénicos en hígado y riñón de rata a lo largo del ejercicio.

The evolving concentration of metabolite intermediates for gluconeogenesis in liver and kidney has been studied in rats after varying periods of exercise (swimming in water at 22 degrees C). Lactate consumption by liver, according to the results, does not take place by gluconeogenesis primarily, since the values for malate, aspartate and PEP show a low in vivo activity for phosphoenolpyruvate carboxykinase. The PEP/aspartate ratio, on the contrary, gradually rises in kidney, suggesting a gradual increase in the in vivo activity of phosphoenolpyruvate carboxykinase, which agree quite well with the results of previously obtained in vitro. The prevention of metabolic acidosis by bicarbonate administration affects the metabolite profile in liver during exercise only very slightly. Renal phosphoenol-pyruvate carboxykinase activity in vivo decreases in relation to previously untreated rats, as well as gluconeogenesis, although to a lesser extent.

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.

Influence of the mother's weight and socioeconomic status on the fatty acid composition of human milk.

The influences of the maternal diet and of the mothers' nutritional and socioeconomic status on the fatty acid composition of human milk have not yet been fully elucidated. Fatty acids from capric (8:0) to docosahexaenoic (22:6w3) acids were determined in 209 samples of human milk obtained from voluntary donors. Samples were classified according to the time of lactation and in relation to the socioeconomic status and to the weight of the lactating women. Capric and lauric acid increased in mature milk while palmitic and stearic acids decreased. Polyunsaturated fatty acids (PUFA) of the w6 and w3 series with more than 18 carbon atoms also decreased from colostrum to mature milk. However, the ratio w3 PUFA/w6 PUFA remained unaltered. Oleic acid decreased in milk from mothers of a medium and low socioeconomic status who consumed almost exclusively seed vegetable oils. The mother's weight did not influence the fatty acid composition of her milk.