Granular mucosal lymphocytes in porcine small intestine

A subpopulation of purified, interepithelial lymphocytes from porcine small intestinal mucosa contained cytoplasmic granules. Toluidine blue staining revealed metachromatic granules in 13.64% (606/4,450) cells. The cells had scant organelles, a single large nucleus with obvious invagination of the nuclear membrane, and prominent chromatin. Each cell contained 1 to 10 cytoplasmic membrane-bound granules, 0.6 to 1.5 micron in diameter. These findings indicated that the granular mucosal lymphocytes are related morphologically to mucosal mast cells. The presence of serotonin in the granules, confirmed by the serotonin releasing test, provided functional evidence that granular mucosal lymphocytes are related to mucosal mast cells.     

Effect of heat-stable enterotoxin of Escherichia coli and theophylline on ion transport in porcine small intestine.

The effect of a heat-stable enterotoxin of Escherichia coli was compared with that of theophylline on ion transport in the pig jejunum, using both in vivo and in vitro techniques. The maximal electrical response to heat-stable enterotoxin was only one-half that of theophylline even though the magnitude of the net secretory response was similar. A net, active secretion of HCO3 was partially responsible for the secretory response induced by heat-stable enterotoxin, whereas theophylline induced an active secretion of chlorine which could account for the entire secretory response. Heat-stable enterotoxin elevated tissue cyclic guanosine monophosphate levels, whereas theophylline elevated both cyclic adenosine monophosphate and cyclic guanosine monophosphate. Cyclic guanosine monophosphate levels induced by heat-stable enterotoxin were markedly potentiated by theophylline. Results suggest that HCO3 secretion in the pig jejunum may be controlled by the cyclic guanosine monophosphate system and this system also activates a neutral secretory process which at high heat-stable enterotoxin doses accounts for the bulk of the net secretion observed. Conversely, the chlorine secretion elicited by theophylline is entirely electrogenic and is consistent with results obtained in other species.     

Temporal lectin histochemical characterization of porcine small intestine.

A variety of biotinylated lectins was applied to formalin-fixed intestinal sections from isolator-reared pigs ranging in age from newborn through 12 weeks. Lectin binding to brush borders of villus enterocytes, crypt enterocytes, and dome epithelium, and lectin reactivity within goblet cells and Brunner's glands was semiquantified by microscopy and was used to estimate temporal changes in complex carbohydrates of enteric epithelium. Although variability in binding scores often was observed among pigs of the same age, several general patterns of lectin binding were detected. Dolichos biflorus and Ulex europaeus lectins had increasing binding to brush border membranes as pigs aged. The Dolichos biflorus, however, had decreased binding at the 12-week time point. Neuraminidase-treated Arachis hypogaea and Triticum vulgaris were associated with high mean binding scores at all time points. Canavalia ensiformis bound, with high mean score at all time points, to villus but not to crypt enterocytes. Arachis hypogaea was associated with variable but often high binding scores, regardless of pig age. Succinylated wheat germ agglutinin bound more to crypt than to villus enterocytes. Goblet cells were generally less reactive than were corresponding villi and crypts. Dome epithelium reactivity varied with the lectin used, whereas Brunner's glands reacted with all lectins tested. We conclude that age and regional variations in lectin binding may reflect differences in intestinal function and differentiation. Because complex carbohydrates may act as cell surface receptors for a variety of enteric pathogens, our results indicate that these differences may be partially responsible for age and anatomic differences in susceptibility or resistance to enteric disease.     

Molecular characterisation of fructose transport in equine small intestine

REASONS FOR PERFORMING STUDY: Fructose can be a suitable carbohydrate supplement for horses before and/or during endurance exercise. In comparison to glucose, the ingestion of fructose results in a lower insulin peak and less marked fluctuations in blood glucose during exercise, potentially avoiding hypoglycaemia-induced exhaustion. OBJECTIVES: To assess the capacity of the equine small intestine to absorb fructose and to determine the mechanism, molecular structure and properties of equine intestinal fructose transport. METHODS: Using PCR-based strategies, RNA isolated from equine small intestine and primers designed to homologous regions of the fructose transporter, GLUT5, cDNA of other species, we cloned and sequenced equine GLUT5 (eGLUT5). Northern and western blot analyses, in conjunction with immunohistochemistry, utilising eGLUT5 cDNA and antibodies, assessed expression of eGLUT5 along the longitudinal and radial axes of the small intestine. Functional properties of fructose transport in intestinal brush-border membrane vesicles were measured using the rapid-filtration technique. RESULTS: eGLUT5 is expressed in the villus enterocytes with highest levels in duodenum>jejunum and lowest in the ileum. Kinetic studies indicate eGLUT5 is a low affinity, high capacity transporter. CONCLUSIONS: Equine small intestine has the capacity to absorb fructose. POTENTIAL RELEVANCE: The molecular probes produced in these studies can be used as diagnostic aids to determine equine intestinal monosaccharide malabsorption.     

Lectin histochemical characterisation of the porcine small intestine around weaning

The present study was undertaken to characterise the carbohydrate profile of the porcine small intestine using lectin histochemistry during the period from 3 days prior to weaning to 9 days post-weaning. A total of 56 piglets weaned at 4 weeks of age were included in the experiment. The most prominent changes in the glycosylation pattern were observed in the goblet cells. The highest lectin reactivity of the goblet cells in the crypts was observed 7 days post-weaning which suggests that the protective effect of the mucus layer against pathogenic bacteria is increasing during the postweaning period. The staining pattern of the apical membrane remained unchanged during the experimental period. This indicates that the glycosylation process in the goblet cells is rapidly inducible whereas changes in the glycosylation pattern of the apical membrane requires more time. The glycosylation pattern of both goblet cells and apical membrane differed between the positions of the small intestine. As glycoconjugates can act as attachment sites for microorganisms, these differences in the distribution of sugar residues may be one explanation for the site-specificity of certain pathogens.     

In vitro response to noradrenaline of small intestine taken from normal and grass sickness-affected horses

Small intestine was taken from the caudal flexure of the duodenum and the terminal ileum proximal to the ileocaecal fold of 25 horses, 9 with acute grass sickness (AGS), 12 with subacute grass sickness (SAGS) and 12 with chronic grass sickness (CGS). The motility in the samples was measured isometrically either within 1 h of death or after storage for 24 h at 4°C.In control tissue, noradrenaline produced contractions of muscle strips which did not involve a muscarinic cholinergic mechanism and which were unaffected by the 1 antagonist prazosin but were blocked by the 2 antagonist yohimbine. Pretreatment with the antagonist phentolamine prevented the contractile response to noradrenaline and the background contractions either continued at a reduced rate and amplitude or were abolished after a few minutes. Thus, following blockade, noradrenaline reduced the background contraction rate by an effect on inhibitory adrenoceptors. The rate of background contractions in duodenal preparations was significantly greater than that in control ileal preparations.Although cold storage for 24 h caused a reduction in the background contraction rates of the control preparations, there was no effect on the contractile responses to noradrenaline, the associated pharmacology being similar to that of fresh tissue. This suggests that noradrenaline-evoked contraction was not dependent on enteric neural elements.The response to noradrenaline by grass sickness-affected tissue was generally similar to that of tissue from control horses, with an immediate contraction which was 2 sensitive. The contractile response to noradrenaline after propranolol was significantly reduced in the CGS group and there were significant differences between the AGS, CGS and control groups. There was a significant difference between the ileal preparations from the control and SAGS groups in their response to noradrenaline following pretreatment with propranolol.     

Identification of methionine and lysine transport systems in cattle small intestine

Incubations of bovine ileal mucosa were used to identify the amino acid transport systems responsible for methionine and lysine uptake in cattle small intestine. Uptake was expressed as micromole per milliliter of cell water. Lysine uptake was shown to be sodium (Na) independent. Specific transport components of lysine uptake were estimated to be: 63% occurring through the cationic (Y+) system, 17% via the neutral alanine (A) and alanine, serine, cysteine (ASC) amino acid transport systems, and 20% through nonsaturable processes. Physiological concentrations of competitive inhibitors altered (P less than .05) lysine uptake. Methionine uptake was shown to occur by both Na-independent and Na-dependent transport systems. The specific transport components of methionine uptake were estimated as 54% occurring through the neutral branched chain leucine (L) systems, 28% through the ASC system, 15% through the A system and 25% through nonsaturable processes. Physiological concentrations of competitive inhibitors did not inhibit (P greater than .05) methionine uptake.     

Methionine and lysine uptake by cattle small intestine in vitro

Incubations of bovine intestinal mucosa were characterized for location of maximal uptake of amino acid, steady-state accumulation time of the amino acids, methionine and lysine. Photomicrographs validated that cattle intestinal mucosa was isolated by the technique described. Methionine and lysine uptake was expressed as micromoles of amino acid per milliliter of cell water. The most active site of methionine and lysine uptake was the mid-ileum, while the proximal jejunum was the least active site. Both methionine and lysine uptake showed high correlation relative to intestinal region. Methionine uptake was shown to be saturated with concentrations greater than 1 mM. Lysine transport appeared to be approaching saturation at concentrations between .5 mM to 1 mM, revealing that cattle intestinal mucosa has a lower transport capacity for lysine than methionine. These results indicate the usefulness of a technique through which substrate uptake (amino acid, carbohydrate, etc.) can be characterized in cattle small intestine.     

In vitro characterization of porcine juvenile articular cartilage

Dispersed cell cultures were established from the articular cartilage of the proximal portion of the humerus of young pigs. Articular and epiphyseal portions of the cartilage were separated, minced, and enzymatically dispersed, using bacterial collagenase. Morphologically, 2 cell types were observed, using phase-contrast microscopy. Smaller polygonal cells (32.5 +/- 3.5 microns diameter) containing cytoplasmic granules were found in both areas of the cartilage. In cultures from the articular region, cells grew as monolayer cultures and initially did not demonstrate contact inhibition. In cultures from the epiphyseal region, cells grew in a multilayered manner in a colonial arrangement with cells being released from the center of the colony into the culture medium. Small granular particles (0.03 to 0.08 micron diameter) were secreted by cells in both culture systems. Particle secretion was greater in epiphyseal cultures than in articular cultures with the rate decreasing as confluency was approached. These particles stained positively for lipid and alkaline phosphatase. Acridine orange was also incorporated into the granules. The 2nd cell type, a stellate-shaped cell (60 +/- 7.6 micron diameter), was found mainly surrounding the outside of colonial areas in epiphyseal cultures. These cells did not secrete small granular particles and stained positive for factor VIII. Evaluation of cultures by scanning and transmission electron microscopy further supported the presence of 2 cell types. With scanning electron microscopy, the smaller polygonal cell was characterized by varying sizes of blebs (0.03 to 0.1 micron diameter) associated with the cell membrane and small cytoplasmic processes projecting from the cell's surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro activity of tiamulin against porcine mycoplasmas

The activity of tiamulin against Mycoplasma hyopneumoniae, M flocculare, M hyorhinis and M hyosynoviae grown in liquid medium was assessed in vitro. With the first three of these mycoplasmas, the activity of tylosin and oxytetracycline was observed in parallel. Tiamulin was more active against M hyopneumoniae and M flocculare, but there was less disparity between the three antibiotics with the strain of M hyorhinis tested. Tiamulin was notably more active against M hyosynoviae than against M hyopneumoniae. It was more difficult to suppress M hyopneumoniae than the other mycoplasmas with tiamulin. This persistence of M hyopneumoniae was more striking when M hyopneumoniae and M hyosynoviae were tested in parallel.     

The early postnatal pattern of vesicle formation in different regions of the porcine small intestine

In the early postnatal period, the permeability of the piglet small intestine is high to compensate for the absence of trans-placental transfer of immunoglobulins during the fetal period. Vesicles, which mainly reflect the uptake of macromolecules and other colostral/milk components, were studied in three different regions of the small intestine – proximal, mid and distal – in a total of twelve piglets on day 0 (unsuckled and colostrums-fed), 2 and 6 (all suckled). Tissues were sampled and prepared for light microscopy (paraffin and cryo) and trans-electron microscopy. Different methods were applied to visualize cytoplasmatic subcellular components such as fat (Oil red O) and carbohydrates (PAS). Appearance and morphology of the epithelial vesicles were compared.

In the proximal region several small supranuclear and a single large subnuclear electron dense, eosinophilic and PAS+ vesicle were present. They disappeared after 2 days (gut closure) and on day 6 adult-looking epithelial cells were present. In the distal region of day 0 pigs digestion vesicles/flocculent vesicles were observed in the cytoplasma. The vesicles appeared empty but with eosinophilic, PAS+ and electron dense precipitations. The size and variation of these vesicles increased with age. Fat absorption increased markedly from day 2 to day 6.

The observations indicate that in general colostral absorption processes are initiated prenatally, persist after birth and in the distal region also after gut closure. Fat absorption increased after gut closure, and in the distal region the increase correlated with e.g. IgG absorption.     

Effect of simulated transport stress on the rat small intestine: A morphological and gene expression study

The present study investigated the effects of simulated transport stress on morphology and gene expression in the small intestine of laboratory rats. Sprague Dawley rats were subjected to 35 °C and 0.1×g on a constant temperature shaker for physiological, biochemical, morphological and microarray analysis before and after treatment. The treatment induced obvious stress responses with significant decreases in body weight (P < 0.01), increases in rectal temperature, serum corticosterone (CORT), serum glucose (GLU), creatine kinase (CK) and lactate dehydrogenase (LDH) levels (P < 0.01), as well as expression of Hsp27/70/90 mRNA (P < 0.05; P < 0.01). The rat jejunum was severely damaged and apoptotic after mimicking transport stress, which may mainly be related to cell death, oxidation reduction and hormone imbalance determined by microarray analysis. The bioinformatics analysis from the present study would provide insight into the potential mechanisms underlying transport stress-induced injury in the rat small intestine.     

Endogenous prostanoids control ion transport across neonatal porcine ileum in vitro

In contrast to the net absorption of Na and Cl ions observed in vivo, porcine small intestine had a net secretion of these ions in vitro. These discrepancies between in vivo and in vitro results have led to difficulties in interpretation of studies investigating mechanisms of intestinal secretion and diarrhea in this species. To examine the influence of endogenous prostanoids on ion transport in neonatal porcine ileum in vitro, tissues were prepared and studied in indomethacin. Net absorption of Na, reversal of net Cl secretion to net absorption, and decreased short circuit current were observed. Conversely, addition of prostaglandins to indomethacin-treated tissues reversed these effects and reestablished conditions similar to those observed in control tissues. Control tissue was essentially refractory to the effects of exogenous prostaglandins. Results indicate that under in vitro conditions, ion transport in neonatal porcine ileum is tightly regulated by endogenous prostanoids that abolish the neutral NaCl absorptive mechanism and elicit electrogenic Cl secretion. However, concentrations of these prostanoids may have been artificially high as a result of tissue preparation for in vitro study.     

In vitro evaluation of an intraluminal solution to attenuate effects of ischemia and reperfusion in the small intestine of horses

OBJECTIVE: To evaluate the efficacy of intraluminal administration of a customized solution during low-flow ischemia and reperfusion in the jejunum of horses. SAMPLE POPULATION: Segments of jejunum obtained from 13 healthy adult horses. PROCEDURE: In isolated segments of jejunum maintained in an extracorporeal circuit, arterial flow was reduced to 20% of baseline for 40 minutes (ischemia) followed by 60 minutes of reperfusion. In 2 groups, a customized solution (concentrations, 12.5 and 25%, respectively) was placed in the lumen prior to low-flow ischemia and maintained during reperfusion. The control group received intraluminal lactated Ringer's solution for the same duration. Various metabolic, hemodynamic, histologic, and permeability variables were recorded. RESULTS: The 12.5% solution resulted in less histomorphologic injury and reduced mucosal permeability to albumin, compared with the 25% solution and the lactated Ringer's solution. Morphologic injury and permeability were reduced in tissues that received the 25% solution, compared with the control group, but this difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: Use of a 12.5% customized solution appeared to minimize injury in the isolated extracoporeal jejunal loop, which provides some indication that it might be useful in clinical situations.     

Electrophysiological characterization of electrolyte and nutrient transport across the small intestine in horses

The aim of this study was to characterize the transport mechanisms of electrolytes and nutrients across the jejunum of nine healthy horses electrophysiologically. The stripped mucosa was mounted in Ussing chambers and tissue conductances (G_t) and short circuit currents (I_sc) were continuously monitored. After blocking the sodium and potassium channels with amiloride, tetraethylammonium chloride (TEA) and barium, chloride secretion was stimulated by carbachol and forskolin. Subsequently, chloride channels were inhibited by 4,4′‐diisothiocyanato‐stilbene‐2,2′‐disulfonic acid, 5‐nitro‐2‐(3‐phenylpropylamino)benzoic acid, CFTR_inh‐172, N‐(2‐naphtalenyl)‐(3.5‐dibromo‐2.4‐dihydroxyphenyl)methylene glycine hydrazide (GlyH‐101) and glibenclamide and their dose–response effect was investigated. The response to glucose, l ‐alanine and glycyl‐l ‐glutamine was determined at two different mucosal pH values (pH 7.4 and 5.4 respectively). Mean basal I_sc was ?0.47 ± 0.31 μEq/cm²h and mean G_t was 22.17 ± 1.78 mS/cm². Amiloride and TEA did not alter the baseline I_sc. Barium, carbachol and forskolin significantly increased I_sc. Irrespective of the dose, none of the chloride inhibitors changed I_sc. All nutrients induced a significant increase in I_sc with the increase being significantly higher at pH 7.4 than at pH 5.4. In conclusion, there is evidence that chloride secretion in horses may be different from respective transport mechanisms in other species. The glucose absorption is suggestive of a sodium‐dependent glucose cotransporter 1. However, a decrease in luminal pH did not stimulate current response to peptides as shown for other mammals.     

In vitro immunomodulatory effect of nisin on porcine leucocytes

Nisin, a lantibiotic bacteriocin, has been used for years as a natural food preservative. In addition to its antimicrobial activity, nisin also shows immunomodulatory properties, and the nisin‐producing Lactococcus lactis strain has been successfully tested as a probiotic in weaned piglets. However, the impact of nisin on porcine immune cells has not yet been explored. The objective of the present study was to examine the in vitro immunomodulatory effect of nisin on porcine peripheral blood leucocytes. The whole heparinized blood samples or freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated with different nisin concentrations (0, 1.56, 3.125, 6.25, 12.5, 25 or 50 µg/ml) for 1, 24, 48 or 72 hr. Escherichia coli bacteria were used to stimulate blood phagocytes, while concanavalin A and lipopolysaccharide from E. coli were used as mitogens. Control cells remained unstimulated. MTT colorimetric assay was used to evaluate PBMCs viability and mitogenic response. Phagocyte activity and T‐cell proliferation were measured by flow cytometry. Flow cytometer was also used for immunophenotyping of T cells. Cytokine levels in the culture media were determined using commercial immunoassay (ELISA) kits. The highest concentration of nisin exhibited proliferative activity (p ? 0.05), stimulated interleukin‐1 beta (IL‐1β) and interleukin‐6 (IL‐6) production (both at p ? 0.001), and increased the percentage of CD4⁺CD8⁺ T cells (p ? 0.001) among unstimulated leucocytes. After cell stimulation, however, the highest nisin concentration showed antiproliferative activity (p ? 0.05), decreased phagocytic functions (p ? 0.05) and inhibited the synthesis of IL‐6 (time‐ and concentration‐dependent effect). As a typical bacterial product, nisin had a stronger impact on innate immune cells, and its effect on T cells was likely a consequence of the modulation of the activity of antigen‐presenting cells. Nisin may be a good candidate as an immunomodulator in pig breeding.