An LGG-derived protein promotes IgA production through upregulation of APRIL expression in intestinal epithelial cells.

p40, a Lactobacillus rhamnosus GG (LGG)-derived protein, transactivates epidermal growth factor receptor (EGFR) in intestinal epithelial cells, leading to amelioration of intestinal injury and inflammation. To elucidate mechanisms by which p40 regulates mucosal immunity to prevent inflammation, this study aimed to determine the effects and mechanisms of p40 on regulation of a proliferation-inducing ligand (APRIL) expression in intestinal epithelial cells for promoting immunoglobulin A (IgA) production. p40 upregulated April gene expression and protein production in mouse small intestine epithelial (MSIE) cells, which were inhibited by blocking EGFR expression and kinase activity. Enteroids from Egfrfl/fl, but not Egfrfl/fl-Vil-Cre mice with EGFR specifically deleted in intestinal epithelial cells, exhibited increased April gene expression by p40 treatment. p40-conditioned media from MSIE cells increased B-cell class switching to IgA+ cells and IgA production, which was suppressed by APRIL receptor-neutralizing antibodies. Treatment of B cells with p40 did not show any effects on IgA production. p40 treatment increased April gene expression and protein production in small intestinal epithelial cells, fecal IgA levels, IgA+B220+, IgA+CD19+, and IgA+ plasma cells in lamina propria of Egfrfl/fl, but not of Egfrfl/fl-Vil-Cre, mice. Thus p40 upregulates EGFR-dependent APRIL production in intestinal epithelial cells, which may contribute to promoting IgA production.

Neonatal colonization of mice with LGG promotes intestinal development and decreases susceptibility to colitis in adulthood.

Development of the intestinal microbiota during early life serves as a key regulatory stage in establishing the host-microbial relationship. This symbiotic relationship contributes to developing host immunity and maintaining health throughout the life span. This study was to develop an approach to colonize conventionally raised mice with a model probiotic bacterium, Lactobacillus rhamnosus GG (LGG), and to determine the effects of LGG colonization on intestinal development and prevention of colitis in adulthood. LGG colonization in conventionally raised was established by administering LGG to pregnant mice starting at gestational day 18 and pups at postnatal days 1- 5. LGG colonization promoted bodyweight gain and increased diversity and richness of the colonic mucosa-associated microbiota before weaning. Intestinal epithelial cell proliferation, differentiation, tight junction formation, and mucosal IgA production were all significantly enhanced in LGG-colonized mice. Adult mice colonized with LGG showed increased IgA production and decreased susceptibility to intestinal injury and inflammation induced in the dextran sodium sulfate model of colitis. Thus, neonatal colonization of mice with LGG enhances intestinal functional maturation and IgA production and confers lifelong health consequences on protection from intestinal injury and inflammation. This strategy might be applied for benefiting health in the host.

The relationship of oxidative stress, adiposity and metabolic risk factors in healthy Black and White American youth.

UNLABELLED: What is already known about this subject African Americans are disproportionately affected by obesity and other metabolic risk factors in comparison to White Americans. Increasing prevalence of obesity has been associated with concomitant increases in childhood hypertension, dyslipidaemia and type 2 diabetes. Oxidative stress is associated with obesity in both adults and children. What this study adds Oxidative stress is positively associated with total body fat and truncal fat, but not with body mass index (BMI) or BMI z-score in healthy youth. Oxidative stress is associated with diastolic blood pressure in African American but not in White American healthy youth. BACKGROUND: Oxidative stress is elevated in obese youth, but less is known regarding racial disparities in the relationship of oxidative stress with metabolic risk factors. OBJECTIVES: To determine the relationship between oxidative stress and metabolic risk factors, adiposity, leptin, adiponectin and cardiovascular fitness (VO2PEAK ) in healthy African American and White American youth. METHODS: A marker of oxidative stress (F2 -isoprostane), validated markers of metabolic risk factors, fitness and body composition were measured in African American (n = 82) and White American (n = 76) youth (8-17 years old) recruited over a range of BMI percentiles (4th to 99th). RESULTS: F2 -isoprostane concentration was positively correlated with percentage body fat (r = 0.198) and percentage truncal fat (r = 0.173), but was not different between African American and White American males and females (P = 0.208). African American youth had significantly higher mean systolic and diastolic blood pressure (P = 0.023 and P = 0.011, respectively), body weight, BMI percentile and Tanner stage. After adjusting for gender, age, BMI and Tanner stage, African American youth varied from White Americans in the association of F2 -isoprostane with diastolic blood pressure (P = 0.047), but not with systolic blood pressure, triglycerides, VO2PEAK or homeostatic model assessment for insulin resistance (all P > 0.05). CONCLUSIONS: Oxidative stress, as measured by urinary F2 -isoprostane concentrations, was positively associated with percent body fat and truncal fat in youth. Oxidative stress levels were similar among African American and White American youth. Among markers of the metabolic syndrome, a significant difference between African American and White American youth was demonstrated only in the association of oxidative stress with diastolic blood pressure.

Effect of dairy and non-dairy calcium on fecal fat excretion in lactose digester and maldigester obese adults.

BACKGROUND: The effect of dietary calcium (Ca) on fecal fat excretion in lactose maldigestion is not known. OBJECTIVE: To investigate the effect of dairy and non-dairy dietary Ca on fecal fat excretion in lactose digesters and maldigesters during moderate energy restriction. DESIGN: A randomized cross-over trial comparing the effect of 500 mg versus 1500 mg dairy and non-dairy Ca on fecal fat excretion in 34 healthy adults during moderate (-30%) energy restriction induced weight loss for 12 weeks. The participants were classified as lactose digester or maldigester on the basis of breath hydrogen test. MEASUREMENTS: Anthropometric parameters and body composition, resting energy expenditure, energy and nutrient intake, fecal fat, physical activity, blood pressure, blood and urine sampling for pertinent measurements. RESULTS: Fecal fat loss expressed as percent of fat intake was significantly higher with 1500 mg (high Ca) as compared with 500 mg (low Ca) Ca intake per day (mean: 3.0%; 95% CI: 2.3 to 3.7%; P<0.001) independent of Ca source and lactose digestion status. CONCLUSIONS: During a moderate energy restriction induced weight loss, a high-Ca diet causes an increase in fecal fat excretion independent of Ca source. Ca intake related fecal fat loss is also independent of the ability to digest lactose and it is not diminished over time (US Clinical Trial Registration: Clinicaltrials.gov NCT00808275).

Patterns of physical activity in free-living adults in the Southern United States.

OBJECTIVE: To examine the relationship between the amount and patterns of physical activity (PA), body fatness, and age in a heterogeneous adult population in the free living. DESIGN: Cross-sectional study of the amount of PA over a 1-week period. The amount of body movements during PA (PA counts*10(3)) and time spent on various PA intensity categories were calculated from a triaxial accelerometer and compared with subject characteristics, including body fat from hydrodensitometry. PARTICIPANTS: Adult healthy men (n=48) and women (n=72) were recruited from the Nashville, Tennessee area and their PA was monitored in their free-living environment. RESULTS: The average weekday PA counts (176.5+/-60.3, P=0.002, r(2)=0.294), PA counts day-to-day variability (47.3+/-32.7, P=0.002, r(2)=0.286), daily maximum PA counts (241.9+/-89.2, P=0.001, r(2)=0.327), minute-to-minute variability on weekdays (0.281+/-0.091, P=0.001, r(2)=0.362), and the difference between maximum and minimum daily PA counts (130.6+/-78.3, P=0.008, r(2)=0.243) were significantly and negatively correlated with body fatness. During awake time, both men and women spent 10-12 h on low intensity (1.0-2.9 metabolic equivalents (METs)) PA, approximately 1 h on moderate (3.0-5.9 MET), and less than 10 min on vigorous (>6.0 MET) PA each day. On weekends, men and women spent more time at rest (1 MET), less time on low-intensity PA, and men spent more time on moderate PA than on weekdays. CONCLUSIONS: In adults living in the Southern US the amount of free-living PA was negatively correlated with body fatness. Both men and women spent the majority of active time on low and moderate PA. PA patterns on weekends were different than on weekdays and were related to sex and age, but not to body fatness. SPONSORSHIP: National Institutes of Health, US.

Energy expenditure determined by self-reported physical activity is related to body fatness.

OBJECTIVE: Activity self-reports are a commonly used tool in assessing daily physical activity (PA) and associated energy expenditure (EE). This study examined the effect of relative body fatness (%BF) on differences between self-reported and measured duration and associated EE in healthy adults. RESEARCH METHODS AND PROCEDURES: Men and women (n = 115, age 38+/-9 years), ranging in %BF from 7.9% to 58.9%, spent two separate days (normal and exercise) in a whole-room indirect calorimeter where EE was measured. While in the room calorimeter, subjects reported the type, intensity, and duration of each performed PA. The Compendium of Physical Activity was used to calculate the energy cost of each reported activity. The EE of all self-reported activities (EEr) was categorized into four intensity levels, synchronized, and compared with EE from the room calorimeter (EEm). RESULTS: With increasing %BF, subjects significantly overestimated duration of more strenuous activities (> or =4.5), while underestimating moderate activities (2.5 to 4.4 metabolic equivalents (METs)). Misreporting of duration and/or intensity caused an overestimation or underestimation of PA-associated EE at these levels. Reported EE sleep was lower than measured EE sleep, although both had similar durations. As a result, total EEr was similar to EEm. DISCUSSION: Individual variability of daily total PA and associated EE generated from self-reports in adults is high. Persons with a higher %BF report duration and/or intensity of moderate to high levels of PA with lower accuracy than leaner individuals. We conclude using the Compendium of Physical Activity is not suitable for the accurate estimation of self-reported EE of AA in adults with a higher %BF.

Increased intestinal epithelial proliferation in metallothioneine-transforming growth factor alpha transgenic mice.

BACKGROUND AND AIM: The epidermal growth factor (EGF) peptide family includes six closely-related proteins, all of which bind to the EGF receptor. In the intestinal epithelium, transforming growth factor alpha (TGFalpha) appears to be the most physiological ligand for the EGF receptor. The present studies were designed to examine the effect of TGFalpha overexpression on duodenal epithelial proliferation using a metallothioneine-inducible promoter/enhancer transgenic mouse (MT-TGFalpha). The MT-TGFalpha mouse model was further studied to determine the in vivo effect of unregulated TGFalpha production on the physiological proliferative responses to fasting and refeeding. METHODS: MT-TGFalpha mice were given 25 mM oral ZnSO4 to induce transgene expression and were studied 1 to 2 months later. Duodenal histology was analyzed morphometrically in well-oriented transverse sections. The vincristine metaphase-arrest technique was used to assess proliferation in duodenal crypts. Immunohistochemical staining and in situ hybridization were used to assess transgenic TGFalpha protein and mRNA expression, respectively. RESULTS: Normally fed MT-TGFalpha mice had deeper crypts (0.12 vs. 0.08 mm), longer villi (0.66 vs. 0.54 mm), and greater luminal diameters (2.65 vs. 2.19 mm) compared to controls (P<0.05 for all three dimensions). The crypt cell mitotic index in normally fed transgenic mice was 1.5 fold greater than the index in normally fed controls (20+/-2 vs. 35+/-4 mitoses per crypt; P <0.05). Fasting and refeeding MT-TGFalpha mice resulted in no significant change in their high baseline rate of crypt proliferation, while proliferation in control mice rose from a lower baseline during fasting to a level with refeeding that approximated rates in MT-TGFalpha mice. Transgenic TGFalpha protein and mRNA were localized to the villus epithelial compartment with little or no evidence of mRNA or protein expression in the crypt epithelium. CONCLUSION: Overproduction of TGFalpha in the mouse duodenal epithelium results in a pronounced increase in crypt epithelial cell proliferation and a resulting increase in the dimension of the crypt/villus unit. This appears to be mediated through a paracrine and/or juxtacrine effect on crypt cells by TGFalpha produced in the villus epithelium. Fasting and refeeding experiments suggest that TGFalpha may also play a role in the proliferative response to refeeding or that the full potential for proliferation is realized by TGFalpha overexpression alone. Collectively, these studies suggest that TGFalpha is a physiological autocrine and paracrine regulator of small intestinal epithelial proliferation.

Electrolyte fluxes in the gut and oral rehydration solutions.

This article provides a brief overview of the normal physiology of water and electrolyte fluxes across the gut as a prerequisite for understanding the pathologic disturbances occurring with diarrheal illnesses. In turn, the rationale for the use of oral rehydration solutions in diarrheal disorders is explored.

Effect of burn injury on intestinal Na+/H+ exchangers.

The Na+/H+ exchangers at the brush border membrane (BBM) and the basolateral membrane (BLM) are each distinct with different kinetic and pharmacologic characteristics. At the BBM, Na+/H+ exchange provides an acid microenvironment necessary for nutrient transport. At the BLM, the Na+/H+ exchanger regulates intracellular pH and cell volume which affect cell growth and repair. This study was designed to determine the effect of burn injury on Na+/H+ exchange at the BBM and BLM of the rat enterocyte. Adolescent Sprague-Dawley rats were divided into control (n = 6) and 20% scald burn groups (n = 6). Using differential centrifugation and percoll density gradient techniques, BBM and BLM vesicles were prepared from the jejunum. 22Na+ uptake was measured using a rapid filtration technique. Initial rate uptake studies showed that the slope of Na+ uptake in BBM (y = 0.06x + 0.12, r2 = 0.99) and BLM (y = 0.075x + 0.1, r2 = 0.99) of the control group was higher (P < 0.05) than that in the burn group (y = 0.0345x + 0.06, r2 = 0.98 and y = 0.056x + 0.01, r2 = 0.99). To determine whether the changes in transport are related to the feeding pattern in burn rats, similar experiments were done in pair-fed animals. The initial rate uptake studies of BBM showed a similarily greater slope of Na+ uptake in pair-fed control animals compared to the burn group (y = 0.043x + 0.06, r2 = 0.99 and y = 0.062x + 0.008, r2 = 0.98; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal Na+/H+ exchanger activity is up-regulated by bowel resection in the weanling rat.

Increased Na+/H+ exchanger activity is associated with cellular hyperplasia. Cellular hyperplasia is an adaptive response to small-intestinal resection. Therefore, we hypothesized that the small-intestinal Na+/H+ exchanger activity increases in response to small-intestinal resection. Twenty-one-d-old, male Sprague-Dawley rats were randomly divided to receive either a 70% small intestinal resection (n = 59), or a mid-small intestinal transection (n = 49). Seven d postoperatively, the animals were killed and the Na+/H+ exchanger activity of the intestinal remnants was studied by a well validated brush border membrane vesicle technique. The initial rate of Na+ uptake in the presence of an outwardly directed pH gradient and the Vmax of the amiloride-sensitive Na+ uptake were significantly increased (p < 0.01 and p < 0.001, respectively) in the resection as compared with the transection remnants and to a greater magnitude in the distal as compared with the proximal remnants. Km values were not significantly different. The amiloride-sensitive Na+ uptake in the setting of various intravesicular pH was significantly greater (p < 0.001) in the distal resection as compared with the distal transection remnants, with points of enhanced Na+/H+ exchanger activity of intravesicular pH 6.62 and 6.87, respectively. The presence and activation of the Na+/H+ exchanger's internal modifier site was confirmed by demonstrating the effect of intravesicular pH on Na+ efflux. The present study demonstrates an up-regulation of intestinal Na+/H+ exchange activity in a small-bowel resection model in the weanling rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a distinct Na(+)-H+ exchanger in basolateral membranes of human jejunum.

Kinetically distinct Na(+)-H+ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na(+)-H+ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na(+)-H+ exchanger may function in homeostatic intravesicular pH (pHi) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na(+)-H+ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H+ gradient [pHi/extravesicular pH (pHo) = 5.2/7.5] resulted in a Na+ uptake overshoot (1.45 +/- 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 +/- 0.13 nmol/mg protein). Na+ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na+ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K+ and the ionophore valinomycin. Na+ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r2 = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID50 value for amiloride of 29 microM (fourfold lower than ID50 for brush-border Na(+)-H+ exchanger). Kinetic studies of amiloride-sensitive Na+ uptake revealed a maximal velocity = 1.53 +/- 0.19 nmol.mg protein-1.5 s-1 and Michaelis constant = 9.83 +/- 3.5 mM. By varying pHi a sigmoidal effect of internal H+ on Na+ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pHi on Na+ efflux and Na(+)-Na+ exchange was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.

Increased Na(+)-H+ exchange in jejunal brush border membrane vesicles of spontaneously hypertensive rats.

The spontaneously hypertensive rats and their genetically matched controls, Wistar-Kyoto, serve as models of essential hypertension. The present study was undertaken to determine whether brush border membrane vesicles obtained from jejunal enterocytes of spontaneously hypertensive rats show increased Na(+)-H+ exchange as part of a generalized membrane disorder. Brush border membrane vesicles were prepared from the jejunum of adult spontaneously hypertensive rats and Wistar-Kyoto rats using an Mg2+/ethylene glycol tetraacetic acid precipitation method. Uptake of 22Na by these vesicles was found to be into an osmotically sensitive intravesicular space rather than mere binding. Initial Na+ uptake by brush border membrane vesicles was greater in spontaneously hypertensive rats than in Wistar-Kyoto rats (P less than 0.05). Higher total and amiloride-sensitive Na+ uptake in spontaneously hypertensive rats occurred in the presence of an outwardly directed pH gradient, and uptake became statistically similar to that of Wistar-Kyoto rats in the absence of a pH gradient. Moreover, amiloride-insensitive Na+ uptake under an outwardly directed pH gradient did not differ significantly between the two groups. The enhanced Na(+)-H+ activity in spontaneously hypertensive rats is not due to altered membrane permeability to protons, as is shown by acridine orange-quenching studies. Kinetic studies for amiloride-sensitive Na+ uptake showed a greater Vmax in spontaneously hypertensive rats compared with Wistar-Kyoto rats (1.46 +/- 0.05 vs. 1.08 +/- 0.08 nmol.mg protein-1.7 s-1) but the Km values were similar in the two groups. These finding, along with similar findings previously reported in vascular smooth muscle and renal tissue of SHR, strongly suggest that an increased Na(+)-H+ exchange is related to the development of hypertension.

Methods of investigating intestinal transport.

This article provides a brief overview of well-established in vivo and in vitro methods that have contributed the most to the understanding of transport processes across the gastrointestinal epithelium. In vivo perfusion techniques in humans revolve around double- and triple-lumen per oral tubes. In animals, in vivo techniques include the single and recirculation perfusion techniques and the double-isotope technique for measurement of net absorption. In vitro methods of studying intestinal transport include the everted gut sac technique, the Ussing chamber, the use of isolated epithelial cells, and the use of brush border and basolateral membranes isolated from enterocytes. The use of fluorescent probes for the measurement of intracellular ionic concentrations is a new and powerful in vivo technique that is now being applied to the gastrointestinal tract.

Ontogeny of the Na(+)-H+ exchanger in rat ileal basolateral membrane vesicles.

This study was designed to examine the activity of the Na(+)-H+ exchanger across the basolateral membranes of the ileal enterocyte and its developmental pattern. The function of the Na(+)-H+ exchanger was studied using a well validated basolateral membrane vesicle technique. Na+ uptake represented transport into the vesicle rather than binding as validated by initial rate studies. Na+ uptake represented an electroneutral process as shown by studies in which negative membrane potential was induced by the ionophore valinomycin. Various outwardly directed pH gradients significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups. However, the magnitude of stimulation was significantly different between the age groups with more marked stimulation of amiloride-sensitive Na+ uptake occurring in adolescent rats as compared to weanling or suckling rats. The amiloride sensitivity of the pH stimulated Na+ uptake was investigated using [Amiloride] = 10(-2)-10(-5) M at pHi/pHo = 5.2/7.5. At 10(-2) M amiloride concentration, Na+ uptake was inhibited by 80%, 70%, 77%, in the basolateral membranes of adolescent, weanling and suckling rats, respectively. Dixon plot analysis in both adolescent and weanling rats was consistent with two amiloride binding sites, a low affinity system and a high affinity system. In the suckling rat, on the other hand, the data supported a single high affinity binding site. Kinetic studies revealed a Km for amiloride-sensitive Na+ uptake of 12.6 +/- 6.6, 10.2 +/- 1.77, 9.46 and Vmax of 4.83 +/- 1.22, 4.47 +/- 0.36 and 8.08 +/- 1.92 n.mol.mg.protein-1.7 s-1 in suckling, weanling and adolescent rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)