Elevated Fecal Calprotectin Accompanied by Intestinal Neutrophil Infiltration and Goblet Cell Hyperplasia in a Murine Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system caused by myelin-specific autoreactive T cells. We previously demonstrated intestinal barrier disruption and signs of inflammation in experimental autoimmune encephalomyelitis (EAE), a model of MS. Fecal calprotectin is a disease activity biomarker in inflammatory bowel diseases, released by neutrophils in response to inflammation. We aimed to further investigate EAE manifestations in the gastrointestinal tract and to determine whether calprotectin is a useful biomarker of intestinal inflammation in EAE. Calprotectin was analyzed in feces, cecal contents, and plasma of EAE mice. Infiltrating neutrophils and goblet cells were investigated in different parts of the gastrointestinal tract before the onset of neurological symptoms and during established disease. We found increased calprotectin levels in feces, cecal content, and plasma preceding EAE onset that further escalated during disease progression. Increased neutrophil infiltration in the intestinal tissue concomitant with IL-17 expression and myeloperoxidase activity was found to correlate well with clinical activity. Increased goblet cells in the intestine, similar to irritable bowel syndrome (IBS), were also observed. The results suggest calprotectin as a good biomarker of gastrointestinal inflammation in EAE and the potential of this model as a useful animal model for IBS.

Exocrine Pancreatic Maturation in Pre-term and Term Piglets Supplemented With Bovine Colostrum.

Pre-term infants have an immature digestive system predisposing to short- and long-term complications including feeding intolerance, maldigestion and necrotizing enterocolitis (NEC). Optimal feeding strategies are required to promote maturation of the gut including the exocrine pancreas. Little is known about age- and diet-related development of pancreatic exocrine enzymes following pre-term birth. Currently, bovine colostrum supplementation is investigated in clinical trials on pre-term infants. Using pigs as models for infants, we hypothesized that pancreatic enzyme content is (1) immature following pre-term birth, (2) stimulated by early colostrum supplementation, and (3) stimulated by later colostrum fortification. Thus, using piglets as models for infants, we measured trypsin, amylase, lipase and total protein in pancreatic tissue collected from piglets delivered by cesarean section either pre-term (90% gestation) or close to term. Experiment 1:Pre-term and term pigs were compared at birth and 11 days. Experiment 2: Pre-term and term pigs were either enterally supplemented with bovine colostrum or fed total parenteral nutrition for 5 days, followed by exclusive milk feeding until day 26. Experiment 3: Pre-term pigs were fed bovine's milk with or without colostrum fortification until 19 days. The results showed that pancreatic trypsin, amylase and total protein contents were reduced in pre-term vs. term pigs. Trypsin mainly increased with advancing post-conceptional age (2-fold), while amylase was affected predominantly by advancing post-natal age, and mostly in pre-term pigs from birth to 11 or 26 days. Colostrum feeding in both term and pre-term piglets decreased trypsin and increased amylase contents. Lipase activity decreased with advancing gestational age at birth and post-natal age, with no consistent responses to colostrum feeding, with lipase activities decreasing relative to total pancreatic protein content. In summary, key pancreatic enzymes, amylase and trypsin, are immature following pre-term birth, potentially contributing to reduced digestive capacity in pre-term neonates. Rapid post-natal increases occurs within few weeks of pre-term birth, partly stimulated by enteral colostrum intake, reflecting a marked adaptation capacity. Alternatively, lipase is less affected by pre-/post-natal age and feeding. Thus, there is a highly enzyme-specific and asymmetric perinatal development of the exocrine pancreas.

Maternal Immunoglobulins in Infants-Are They More Than Just a Form of Passive Immunity?

In the present review, we highlight the possible "extra-immunological" effects of maternal immunoglobulins (Ig) transferred to the blood circulation of offspring, either via the placenta before birth or via the colostrum/milk across the gut after birth in different mammalian species. Using the newborn pig as a model, since they are naturally born agammaglobulinemic, intravenously (i.v.) infused purified serum Ig rapidly improved the vitality, suckling behavior, and ensured the survival of both preterm and term piglets. In further studies, we found that proper brain development requires i.v. Ig supplementation. Studies have reported on the positive effects of i.v. Ig treatment in children with epilepsy. Moreover, feeding newborn pigs an elementary diet supplemented with Ig improved the gut structure, and recently a positive impact of enteral or parenteral Ig supplementation on the absorption of polyunsaturated fatty acids (PUFAs) was observed in the newborn pig. Summarized, our own results and those found in the literature, indicate the existence of important extra-immune effects of maternal Ig, in addition to the classical protective effects of transferred maternal passive immunity, including effects on the development of the brain, gut, and possibly other organ systems in the neonate. These additional properties of circulating Ig could have an impact on care guidelines for human neonates, especially those born prematurely with low plasma Ig levels.

The Immature Gut Barrier and Its Importance in Establishing Immunity in Newborn Mammals.

The gut is an efficient barrier which protects against the passage of pathogenic microorganisms and potential harmful macromolecules into the body, in addition to its primary function of nutrient digestion and absorption. Contrary to the restricted macromolecular passage in adulthood, enhanced transfer takes place across the intestines during early life, due to the high endocytic capacity of the immature intestinal epithelial cells during the fetal and/or neonatal periods. The timing and extent of this enhanced endocytic capacity is dependent on animal species, with a prominent non-selective intestinal macromolecular transfer in newborn ungulates, e.g., pigs, during the first few days of life, and a selective transfer of mainly immunoglobulin G (IgG), mediated by the FcRn receptor, in suckling rodents, e.g., rats and mice. In primates, maternal IgG is transferred during fetal life via the placenta, and intestinal macromolecular transfer is largely restricted in human neonates. The period of intestinal macromolecular transmission provides passive immune protection through the transfer of IgG antibodies from an immune competent mother; and may even have extra-immune beneficial effects on organ maturation in the offspring. Moreover, intestinal transfer during the fetal/neonatal periods results in increased exposure to microbial and food antigens which are then presented to the underlying immune system, which is both naïve and immature. This likely stimulates the maturation of the immune system and shifts the response toward tolerance induction instead of activation or inflammation, as usually seen in adulthood. Ingestion of mother's milk and the dietary transition to complex food at weaning, as well as the transient changes in the gut microbiota during the neonatal period, are also involved in the resulting immune response. Any disturbances in timing and/or balance of these parallel processes, i.e., intestinal epithelial maturation, luminal microbial colonization and mucosal immune maturation due to, e.g., preterm birth, infection, antibiotic use or nutrient changes during the neonatal period, might affect the establishment of the immune system in the infant. This review will focus on how differing developmental processes in the intestinal epithelium affect the macromolecular passage in different species and the possible impact of such passage on the establishment of immunity during the critical perinatal period in young mammals.

Absorption of Polyunsaturated Fatty Acid (PUFA) Is Related to IgG Blood Levels of Neonatal Pigs during the First 48 Hours Postpartum.

The current study is aimed at highlighting the impact of enterally or parenterally applied immunoglobulins (Igs) on polyunsaturated fatty acid (PUFA) absorption in newborn pigs. Piglets were chosen as the appropriate model since they are born agammaglobulinemic and any effects of Ig addition can thus be easily monitored. Twenty-one, new born piglets were used in the study. Plasma levels of PUFAs, ARA, DHA, and EPA dropped (similarly to that seen in human infants) by between 40 and 50% in newborn, unsuckled piglets fed an infant formula for 48 h. However, piglets fed the same infant formula but supplied with immunoglobulins (Igs) either orally, by feeding piglets with swine or bovine colostrum, or intravenously, by i.u.a. (intraumbilical artery) infusion of swine or human Ig preparations or swine serum, demonstrated improved growth and PUFA levels similar to those observed at birth. The significant positive correlation was found between the body weight gain, as well as levels of ARA and EPA, and plasma immunoglobulins concentration. These results indicate the importance of the presence of Ig in the blood for appropriate absorption of dietary PUFAs and probably other nutrients in newborn piglets. This may have an impact on the dietary guidelines for human neonates, especially those born prematurely with low plasma Ig levels, since PUFAs are important factors for brain development in early life.

Early effects on the intestinal barrier and pancreatic function after enteral stimulation with protease or kidney bean lectin in neonatal rats.

Gut maturation naturally accelerates at weaning in altricial mammalian species, such as the rat. Mimicking this, gut development can also be induced precociously, 3-4 d earlier than it would occur naturally, by enteral exposure to phytohaemagglutinin (PHA), or various proteases. We investigated the early effects of gut provocation on intestinal barrier and pancreatic functions, to get a better understanding of the mechanisms that initiate gut maturation. The effects of oral administration of protease (trypsin) or PHA to 14-d-old suckling rats were studied during 24 h in comparison with water-fed controls. Intestinal in vivo permeability was assessed by oral administration of different-sized marker molecules and measuring their passage into the blood or urine 3 h later. A period of 24 h following oral administration, both PHA and protease provocation stimulated small intestinal (SI) growth and pancreatic secretion, as indicated by decreased pancreatic trypsin and increased luminal enzyme content. Within 1 h of oral administration, both treatments prevented the absorption of macromolecules to blood that was observed in controls. PHA treatment hindered the passage of fluorescein isothiocyanate-dextran (FD) 4 to blood, whereas protease treatment temporarily increased plasma levels of FD4, and the urine lactulose:mannitol ratio, indicating increased intestinal leakiness. Following protease treatment, fluorescence microscopy showed decreased vesicular uptake of FD70 in the proximal SI and increased epithelial fluorescence in the distal SI. In conclusion, PHA and protease differed in their early effects on the intestinal barrier; both exerted a blocking effect on epithelial endocytosis, whereas protease treatment alone temporarily increased epithelial leakiness, which seemed to be confined to the distal SI.

Induction of precocious intestinal maturation in T-cell deficient athymic neonatal rats.

AIM: To investigate whether gut maturation could be induced precociously in an athymic T-cell deficient neonatal rat model. METHODS: Fourteen day-old athymic (nude) rats (NIH-Foxn1rnu) were gavaged with either phytohaemagglutinin - a lectin from red kidney beans (PHA); trypsin - a protease (Prot); or water - vehicle (control) as a single dose on one day or once a day for 3-day. The nude rats were either nurtured by their mothers or cross-fostered by conventional foster dams of the Sprague-Dawley strain from days 3-5 after birth. At 17 d of age, 72 h after administration of the first treatment, intestinal macromolecular permeability was tested in vivo, prior to euthanasia, after which blood and gut organs were sampled. RESULTS: Provocation with both, PHA and protease, resulted in increased gut growth and maturation in nude rat pups independent of nursing. Foetal-type enterocytes were replaced by non-vacuolated adult-type enterocytes in the distal small intestine epithelium. Decreased intestinal macromolecular permeability (gut closure) was observed, with reduced permeability markers (BIgG and BSA, P < 0.001) in circulation. Increased pancreatic function, with an increased trypsin to protein ratio in pancreas homogenates, was observed independent of nursing in the nude pups. Immunostaining showed the presence of a few CD3+-cells in the intestinal mucosa of the nude pups. The number of CD3+-cells remained unaltered by provocation and no differences were observed between the nursing sets. Growth and vitality of the nude pups were dependent on nurturing, since cross-fostering by conventional dams increased their macromolecular absorptive capacity (BSA, P < 0.05), as well as their passive immunity (RIgG, P < 0.05). CONCLUSION: Precocious gut maturation can be induced by enteral provocation in athymic rat pups, similarly to in euthymic pups, thus showing an independence from thymus-derived T-cells.

Experiments suggesting extra-digestive effects of enteral pancreatic amylase and its peptides on glucose homeostasis in a pig model.

The studies presented were designed to highlight the impact of pancreatic enzymes on glycemic control and insulin response. Blood glucose and plasma insulin levels were monitored after intravenous, oral or direct gut glucose tolerance tests (GTT) in 6 pigs with an intact gastrointestinal tract and in 12 pigs following duodenal-jejunal bypass (DJB) surgery. In the intact pigs, pancreatic enzymes (Creon®) given orally 1 h prior to the GTT, lowered the blood glucose levels during the oral and meal GTT and reduced the plasma insulin response during the intravenous and meal GTT. In DJB pigs, blood glucose and plasma insulin levels were higher following glucose loading into the by-passed biliopancreatic limb as compared to that following glucose loading orally or into the common intestinal limb. Infusion of amylase or amylase peptides together with glucose into the biliopancreatic limb lowered blood glucose levels in DJB pigs. These preliminary data suggest new, extra-digestive, actions of enteral pancreatic enzymes - probably amylase or its peptides - on glucose homeostasis, with an reduction in net glucose absorption into the blood and in insulin response. This ability of digestive enzymes (amylase) to reduce post-prandial hyperglycaemia in an insulin-independent manner could aid in preventing the development of obesity and diabetes.

Importance of neonatal immunoglobulin transfer for hippocampal development and behaviour in the newborn pig.

Neurological disorders are among the main clinical problems affecting preterm children and often result in the development of communication and learning disabilities later in life. Several factors are of importance for brain development, however the role of immunoglobulins (passive immunity transfer) has not yet been investigated. Piglets are born agammaglobulinemic, as a result of the lack of transfer of maternal immunoglobulins in utero, thus, they serve as an ideal model to mimic the condition of immunoglobulin deficiency in preterm infants. Thirty six, unsuckled newborn piglets were fed an infant formula or colostrum and supplemented orally or intravenously with either species-specific or foreign immunoglobulin and then compared to both newborn and sow-reared piglets. Two days after the piglets were born behavioural tests (novel recognition and olfactory discrimination of conspecifics scent) were performed, after which the piglets were sacrificed and blood, cerebrospinal fluid and hippocampi samples were collected for analyses. Both parameters of neuronal plasticity (neuronal maturation and synapse-associated proteins) and behavioural test parameters appeared to be improved by the appearance of species-specific porcine immunoglulin in the circulation and cerebrospinal fluid of the piglets. In conclusion, we postulate possible positive clinical effects following intravenous infusion of human immunoglobulin in terms of neuronal plasticity and cognitive function in preterm infants born with low blood immunoglobulin levels.

Correction: Maturation of the Intestinal Epithelial Barrier in Neonatal Rats Coincides with Decreased FcRn Expression, Replacement of Vacuolated Enterocytes and Changed Blimp-1 Expression.

[This corrects the article DOI: 10.1371/journal.pone.0164775.].

Dietary green-plant thylakoids decrease gastric emptying and gut transit, promote changes in the gut microbial flora, but does not cause steatorrhea.

Green-plant thylakoids increase satiety by affecting appetite hormones such as ghrelin, cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1). The objective of this study was to investigate if thylakoids also affect gastrointestinal (GI) passage and microbial composition. To analyse the effects on GI passage, 16 rats were gavage-fed a control or thylakoid-supplemented high-fat diet (HFD) 30 min before receiving Evans blue. Another 16 rats were fed a control HFD or thylakoid HFD for two weeks prior to the intragastric challenge with Evans blue. The amount of Evans blue in the stomach and the distance of migration in the intestines after 30 min were used as a measurement of gastric emptying and intestinal transit. These were reduced by thylakoid supplementation in the acute study, and however not significantly also after the two-week diet study. The second aim of the study was to investigate if thylakoid-supplementation affects the gut microbiota and amount of faecal fat in healthy human volunteers (n = 34) receiving thylakoid or placebo treatments for three months. Microbiota was analysed using 16S rRNA gene sequencing and qPCR, and faecal fat was extracted by dichloromethane. The total bacteria, and specifically the Bacteriodes fragilis group, were increased by thylakoid treatment versus placebo, while thylakoids did not cause steatorrhea. Dietary supplementation with thylakoids thus affects satiety both via appetite hormones and GI fullness, and affects the microbial composition without causing GI adverse effects such as steatorrhea. This suggests thylakoids as a novel agent in prevention and treatment of obesity.

Maturation of the Intestinal Epithelial Barrier in Neonatal Rats Coincides with Decreased FcRn Expression, Replacement of Vacuolated Enterocytes and Changed Blimp-1 Expression.

BACKGROUND: The intestinal barrier is immature in newborn mammals allowing for transfer of bioactive macromolecules, e.g. protecting antibodies, from mother's milk to the blood circulation and in neonatal rodents lasts until weaning. This passage involves the neonatal-Fc-receptor (FcRn) binding IgG in the proximal and highly endocytic vacuolated enterocytes in the distal immature small intestine (SI). Recent studies have suggested an involvement of the transcription factor B-lymphocyte-induced maturation-protein-1 (Blimp-1) in the regulation of SI maturation in mice. Hence, the objective of the present study was to monitor the development of the intestinal barrier function, in relation to Blimp-1 expression during both natural and precociously induced intestinal maturation in rats. RESULTS: During the suckling period IgG plasma levels increased, while after gut closure it temporarily decreased. This corresponded to a high expression of FcRn in the proximal SI epithelium and the presence of vacuolated enterocytes in the distal SI. The immature foetal-type epithelium was replaced after weaning or induced precocious maturation, by an adult-type epithelium with FcRnneg cells in the proximal and by non-vacuolated enterocytes in the distal SI. In parallel to this epithelial shift, Blimp-1 expression decreased in the distal SI. CONCLUSION: The switch from foetal- to adult-type epithelium, with decreased proximal expression of FcRn and distal replacement of vacuolated enterocytes, was concurrent in the two SI regions and could be used for monitoring SI maturation in the rat. The changes in expression of Blimp-1 in the distal SI epithelium followed the maturation pattern.

Dietary thylakoids reduce visceral fat mass and increase expression of genes involved in intestinal fatty acid oxidation in high-fat fed rats.

Thylakoids reduce body weight gain and body fat accumulation in rodents. This study investigated whether an enhanced oxidation of dietary fat-derived fatty acids in the intestine contributes to the thylakoid effects. Male Sprague-Dawley rats were fed a high-fat diet with (n = 8) or without thylakoids (n = 8) for 2 wk. Body weight, food intake, and body fat were measured, and intestinal mucosa was collected and analyzed. Quantitative real-time PCR was used to measure gene expression levels of key enzymes involved in fatty acid transport, fatty acid oxidation, and ketogenesis. Another set of thylakoid-treated (n = 10) and control rats (n = 10) went through indirect calorimetry. In the first experiment, thylakoid-treated rats (n = 8) accumulated 25% less visceral fat than controls. Furthermore, fatty acid translocase (Fat/Cd36), carnitine palmitoyltransferase 1a (Cpt1a), and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) genes were upregulated in the jejunum of the thylakoid-treated group. In the second experiment, thylakoid-treated rats (n = 10) gained 17.5% less weight compared with controls and their respiratory quotient was lower, 0.86 compared with 0.91. Thylakoid-intake resulted in decreased food intake and did not cause steatorrhea. These results suggest that thylakoids stimulated intestinal fatty acid oxidation and ketogenesis, resulting in an increased ability of the intestine to handle dietary fat. The increased fatty acid oxidation and the resulting reduction in food intake may contribute to the reduced fat accumulation in thylakoid-treated animals.

Decreased insulin secretion and glucose clearance in exocrine pancreas-insufficient pigs.

The effect of exocrine pancreatic function on the glucose-mediated insulin response and glucose utilization were studied in an exocrine pancreas-insufficient (EPI) pig model. Five 10-week-old EPI pigs after pancreatic duct ligation and 6 age-matched, non-operated control pigs were used in the study. Blood glucose, plasma insulin and C-peptide concentrations were monitored during meal (MGTT), oral (OGTT) and intravenous (IVGTT) glucose tolerance tests. Upon post-mortem examination, the pancreatic remnants of the EPI pigs showed acinar fibrotic atrophy but normal islets and ß-cell morphology. The EPI pigs displayed increased fasting glucose concentrations compared with control animals (6.4 ± 0.4 versus 4.8 ± 0.1 mmol l(-1) , P < 0.0001) but unchanged insulin concentrations (2.4 ± 0.6 versus 2.1 ± 0.2 pmol l(-1) ). During the OGTT and IVGTT, the EPI pigs showed slower, impaired glucose utilization, with the disruption of a well-timed insulin response. Plasma C-peptide concentrations confirmed the delayed insulin response during the IVGTT in EPI pigs. Oral pancreatic enzyme supplementation (PES) of EPI pigs improved glucose clearance during IVGTT [AUC(glucose) 1295 ± 70 mmol l(-1) × (120 min) in EPI versus 1044 ± 32 mmol l(-1) × (120 min) in EPI + PES, P < 0.0001] without reinforcing the release of insulin [AUC(C-peptide) 14.4 ± 3.8 nmol l(-1) × (120 min) in EPI versus 6.4 ± 1.3 nmol l(-1) × (120 min) in EPI + PES, P < 0.002]. The results suggest the existence of an acino-insular axis regulatory communication. The presence of pancreatic enzymes in the gut facilitates glucose utilization in an insulin-independent manner, indicating the existence of a gut-derived pancreatic enzyme-dependent mechanism involved in peripheral glucose utilization.

Buserelin treatment to rats causes enteric neurodegeneration with moderate effects on CRF-immunoreactive neurons and Enterobacteriaceae in colon, and in acetylcholine-mediated permeability in ileum.

BACKGROUND: The gonadotropin-releasing hormone (GnRH) analog buserelin causes enteric neuronal loss. Acute stress or injection of corticotropin-releasing factor (CRF) affects motility, secretion, and barrier function of the gastrointestinal tract. The aim of the study was to characterize the CRF immunoreactivity in enteric neurons after buserelin treatment, and to evaluate possible effects of enteric neuropathy on gut microbiota, intestinal permeability, and stress response behavior. RESULTS: Sixty rats were given buserelin (20 µg) or saline subcutaneously for 5 days, repeated four times with 3 weeks in-between. At the study end, enteric neuronal density, enteric expression of CRF, gut microbial composition, and plasma levels of adrenocorticotropic hormone (ACTH) and CRF were analyzed. Intestinal permeability was examined in Ussing chambers and the reaction to stressful events was measured by behavior tests. Buserelin treatment reduced the number of neurons along the entire gastrointestinal tract, with increased relative numbers of CRF-immunoreactive submucosal and myenteric neurons in colon (p < 0.05 and p < 0.01, respectively). The overall microbial diversity and relative abundance did not differ between groups, but Enterobacteriaceae was decreased in colon in buserelin-treated rats (p = 0.020). Basal intestinal permeability did not differ between groups, whereas carbachol stimulation increased ileum permeability in controls (p < 0.05), but not in buserelin-treated rats. Buserelin did not affect stress behavior. CONCLUSIONS: Although buserelin treatment leads to enteric neuronal loss along the gastrointestinal tract with an increased percentage of CRF-immunoreactive neurons in colon, the physiology is well preserved, with modest effects on colon microbiota and absence of carbachol-induced permeability in ileum as the only observed changes.

Monitoring changes in plasma levels of pancreatic and intestinal enzymes in a model of pancreatic exocrine insufficiency--induced by pancreatic duct-ligation--in young pigs.

PURPOSE: Plasma levels of pancreatic and intestinal enzymes were measured after pancreatic duct ligation (PDL) to monitor pancreatic exocrine insufficiency (PEI) in a model using young pigs. MATERIAL/METHODS: Five, 6 week-old pigs (10.9±0.2kg), underwent PDL while age-matched, un-operated pigs were used as controls. Plasma levels of immunoreactive cationic trypsinogen (IRCT), amylase, lipase, and diamine oxidase (DAO) activities were analyzed for 48 days after PDL, including 1 week of oral pancreatic enzyme supplementation (PES) with Creon(®). RESULTS: PDL resulted in an arrested body growth and a rapid surge of pancreatic enzymes (IRCT, amylase and lipase) into the plasma. Nine days after PDL, the plasma levels of these pancreatic enzymes had decreased. IRCT then remained below the level in un-operated pigs while amylase only fell below control at 25 days. The intestinally derived marker DAO and plasma protein levels were unaffected by PDL but DAO decreased slightly with time in PEI pigs. One-week of oral PES restored body growth, but had little effect on pancreatic enzyme plasma levels, except for a tendency towards increased DAO. CONCLUSIONS: The study showed that PEI developed within 1-2 weeks after PDL and that only IRCT is a reliable plasma enzyme marker for this. The reduced plasma DAO indicated that PEI also affected the intestines, while PES therapy restored growth of the PDL pigs and slightly increased plasma DAO, suggesting an improved intestinal function.

Pancreatic and pancreatic-like microbial proteases accelerate gut maturation in neonatal rats.

OBJECTIVES: Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. METHODS: Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14-16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. RESULTS: Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. CONCLUSION: Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals.

Structural and functional consequences of buserelin-induced enteric neuropathy in rat.

BACKGROUND: Women treated with gonadotropin-releasing hormone (GnRH) analogs may develop enteric neuropathy and dysmotility. Administration of a GnRH analog to rats leads to similar degenerative neuropathy and ganglioneuritis. The aim of this study on rat was to evaluate the early GnRH-induced enteric neuropathy in terms of distribution of neuronal subpopulations and gastrointestinal (GI) function. METHODS: Forty rats were given the GnRH analog buserelin (20 µg, 1 mg/ml) or saline subcutaneously, once daily for 5 days, followed by 3 weeks of recovery, representing one treatment session. Two weeks after the fourth treatment session, the animals were tested for GI transit time and galactose absorption, and fecal weight and fat content was analyzed. After sacrifice, enteric neuronal subpopulations were analyzed. Blood samples were analyzed for zonulin and antibodies against GnRH and luteinizing hormone, and their receptors. RESULTS: Buserelin treatment transiently increased the body weight after 5 and 9 weeks (p < 0.001). Increased estradiol in plasma and thickened uterine muscle layers indicate high estrogen activity. The numbers of both submucous and myenteric neurons were reduced by 27%-61% in ileum and colon. The relative numbers of neurons containing calcitonin gene-related peptide (CGRP), cocaine- and amphetamine-related transcript (CART), galanin, gastrin-releasing peptide (GRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), serotonin, substance P (SP), vasoactive intestinal peptide (VIP) or vesicular acetylcholine transporter (VAchT), and their nerve fiber density, were unchanged after buserelin treatment, but the relative number of submucous neurons containing somatostatin tended to be increased (p = 0.062). The feces weight decreased in buserelin-treated rats (p < 0.01), whereas feces fat content increased (p < 0.05), compared to control rats. Total GI transit time, galactose absorption, zonulin levels in plasma, and antibody titers in serum were unaffected by buserelin treatment. CONCLUSIONS: A marked enteric neuronal loss with modest effects on GI function is found after buserelin treatment. Increased feces fat content is suggested an early sign of dysfunction.

Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.