[Modulation of the intestinal microbiota by nutritional interventions]. / Modulation der intestinalen Mikrobiota durch Ernährungsinterventionen.

Humans live in symbiosis with billions of commensal bacteria. The so-called microbiota live on different biological interfaces such as the skin, the urogenital tract and the gastrointestinal tract. Commensal bacteria replace potentially pathogenic microbes, synthesize vitamins and ferment dietary fibre. An imbalance in the bacterial composition of the intestinal microbiota has been associated with various diseases including gut-associated disorders such as inflammatory bowel diseases, colorectal cancer and nonalcoholic fatty liver disease. Furthermore, a shift in the microbiota composition appears to be of pathophysiological relevance which renders the specific modulation of the intestinal microbiota a promising approach in the treatment of the above mentioned diseases. Our intestinal microbiota composition is mainly modulated by dietary macro- and micronutrients but also by secondary plant compounds and synthetic food additives such as emulsifiers and artificial sweeteners. Nutritional interventions with the purpose to modulate the intestinal microbiota show only limited therapeutic potential in the treatment of gut-associated disorders, which may be due to individual differences in the intestinal microbiota composition and a lack of specificity. A combination of newly established technical analytic approaches involving a machine-learning algorithm may bridge the currently existing limitations by providing a personalized, highly-specific and consequently therapeutically effective microbiota modulation.

Cyanidin does not affect sulforaphane-mediated Nrf2 induction in cultured human keratinocytes.

There is increasing interest in the gene-regulatory activities of isothiocyanates and flavonoids in human skin. Nrf2 agonists, such as isothiocyanate sulforaphane (SFN), have been shown to promote chemopreventive effects in skin both in vitro and in vivo. Recent data indicate that different secondary plant compounds may either antagonise or enhance SFN-induced Nrf2 activation. We therefore studied the interactions of a flavonoid, cyanidin and the potent Nrf2 inductor SFN in cultured human keratinocytes (HaCaT cells). We observed that cyanidin does not induce the activation of Nrf2 and its target genes, γ-glutamylcysteine synthetase (γGCS), NAD(P)H:quinone oxidoreductase 1 and haem oxygenase-1 in HaCaT cells. Furthermore, SFN-mediated Nrf2 activation and its target gene expression were not further enhanced by the co-application of SFN with cyanidin.

Cellular uptake, stability, visualization by 'Naturstoff reagent A', and multidrug resistance protein 1 gene-regulatory activity of cyanidin in human keratinocytes.

There is increasing interest in the role of anthocyanidins as potential skin protective phytochemicals. However, little is known if and to what extent anthocyanidins are taken up by the human skin. In the present study cellular uptake (as determined by HPLC), stability, and gene-regulatory activity of cyanidin were determined in human HaCaT keratinocytes in culture. Using the fluorescent dye Naturstoff reagent A cyanidin was visualized in order to determine its cellular accumulation via flow cytometry and fluorescence microscopy. Cyanidin was rapidly taken up by HaCaT cells at relatively low concentrations. Following incubation, cellular cyanidin levels decreased time-dependently most likely due to degradation into protocatechuic acid and phloroglucinol aldehyde. Confocal laser scanning microscopy data demonstrated that cyanidin was mainly present in the cytoplasm. Cellular uptake of cyanidin was accompanied by an inhibition of multidrug resistance protein 1 (involved in cellular efflux of flavonoids) mRNA-levels indicating its gene-regulatory activity. Naturstoff reagent A seems to be a promising fluorescent dye to visualize cyanidin in keratinocytes.

Ochratoxin A impairs Nrf2-dependent gene expression in porcine kidney tubulus cells.

The mycotoxin, ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is frequently present in feedstuffs. Ochratoxin A exhibits a wide range of toxic activities including nephrotoxicity. However, the underlying molecular mechanisms of OTA-induced cellular nephrotoxicity have yet not been fully elucidated. Nrf2 is a basic leucine zipper transcriptional activator essential for the coordinated transcriptional induction of antioxidant and xenobiotic metabolizing enzymes in the kidney. Therefore, in the present study, the effects of OTA on the nuclear translocation and transactivation of the transcription factor Nrf2 as well as mRNA levels of Nrf2 target genes including glutathione-S-transferase and gamma-glutamylcysteinyl synthetase have been studied in cultured porcine kidney tubulus cells (LLC-PK1). Nrf2 was induced by sulforaphane, a well-known activator of this transcription factor. Ochratoxin A significantly decreased gamma-glutamylcysteinyl synthetase and glutathione-S-transferase mRNA levels in LLC-PK1 cells. Decreased mRNA levels of gamma-glutamylcysteinyl synthetase and glutathione-S-transferase were accompanied by a lowered nuclear translocation and transactivation of Nrf2. Furthermore, OTA also lowered Nrf2 mRNA levels. Current data indicate that OTA nephrotoxicity may be, at least partly, mediated by an Nrf2-dependent signal transduction pathway.

Dexamethasone impairs hypoxia-inducible factor-1 function.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of alpha- and beta-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1alpha levels in the cytosol of HepG2 cells, while nuclear HIF-1alpha levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

Effects of a six-week intraduodenal supplementation with quercetin on liver lipid metabolism and oxidative stress in peripartal dairy cows.

The purpose of this study was to evaluate possible effects of quercetin (Q) on liver lipid metabolism and antioxidative status in periparturient dairy cows. The periparturient period is associated with enormous metabolic changes for dairy cows. Energy needs for incipient lactation are too high to be balanced by feed intake, leading to negative energy balance and body fat mobilization. It has been estimated that this leads to the development of fatty liver in about 50% of cows, which are at high risk for disease. Furthermore, the antioxidative status of these cows may be impaired. Quercetin is a plant flavonoid having hepatoprotective and antioxidative potential and the ability to reduce liver lipid accumulation in monogastric animals. Little information is available in regard to these effects in ruminants. To prevent microbial Q degradation in the rumen, Q was administered via a duodenal fistula to improve systemic availability. Five cows of the Q-treated group received, daily, 100 mg of quercetin dehydrate/kg BW in a 0.9% sodium chloride solution from d -20 until d 20 relative to calving, whereas 5 control (CTR) cows received only a sodium chloride solution. Blood samples were taken weekly and liver biopsies were performed in wk -4, -2, and 3 relative to calving. Cows treated with Q showed a tendency ( = 0.082) for lower liver fat content compared with CTR cows. Liver glycogen, glutathione concentrations, and relative mRNA abundance of genes related to hepatic lipid metabolism and antioxidative status as well as parameters of antioxidative status in plasma were not affected ( > 0.1) by Q supplementation. In conclusion, liver fat content in dairy cows tended to be reduced by Q supplementation, but potential underlying mechanisms remain unclear because analyzed parameters related to hepatic lipid metabolism and antioxidative defense were not altered by Q supplementation.

Changes in anesthetic sensitivity and glutamate receptors in the aging canine brain.

This study investigated whether the aging process in dogs is associated with an increased sensitivity to inhalation anesthesia and whether age-related changes in glutamate receptors are related to the increased sensitivity. The mean minimum alveolar concentration (MAC) of isoflurane was 1.82 +/- .08% for 2-3 year olds and 1.45 +/- .06% for 11 years olds, indicating that there was an increased potency of isoflurane in the older dogs as compared to the young. These older animals also showed a significant decrease in binding of [3H]glutamate and [3H]dizocilpine ([3H]MK801) to N-methyl-D-aspartate (NMDA) receptors in multiple cortical and hippocampal regions. The density of binding to NMDA receptors in the cortex, using a single concentration of ligand, correlated significantly with individual MAC values. These results demonstrate that dogs experience an increase in anesthetic potency with increased age, similar to humans, and that age-related changes in the NMDA receptor may represent one mechanism underlying this increased sensitivity to anesthesia.

Pattern of neuropsychological deficit at age five years following neonatal unilateral brain injury.

The pattern of language deficit following left-hemisphere brain injury and visual/spatial deficit following right-hemisphere injury in an adult or older child is well recognized, but has been inconsistently reported following presumed neonatal brain injury. Our prospective study of 24 children at age 5 with documented neonatal unilateral brain injury lends support to the theory of hemisphere specialization at the time of birth. Twelve children who had unilateral left-hemisphere lesion were compared to 12 children with unilateral right-hemisphere lesion of similar timing and severity. Relative visual/spatial deficit following right-hemisphere lesion and receptive language deficit following left-hemisphere lesion were identified. Lateralized measures of grip strength, fine motor speed, and fine motor dexterity were not significantly different between the groups for either hand in this nonhemiparetic study sample. Only one child with a left-hemisphere lesion was left-handed, and only one child (right-lesion) had a hemiparesis.

Sex differences in school-aged children with pervasive attention deficit hyperactivity disorder.

This study investigated 54 children (37 boys and 17 girls) with cross-situational attention deficit hyperactivity disorder (ADHD) to determine whether there are sex differences in the expression of either the primary or secondary symptomatology of ADHD. Results indicated that the male and female ADHD groups were strikingly similar on all measures of primary (impulsivity, inattention, and overactivity) and secondary (learning problems, externalizing symptoms, internalizing symptoms, peer relationship difficulties, and self-perceptions) symptomatology included in this study. The lack of significant sex differences conflicts with prior reports in the literature, and these conflicting results are discussed in terms of differences in inclusion criteria. Implications for understanding the long-term outcome of ADHD in girls are also discussed.

Nephelometric determination of serum amylase and lipase in naturally occurring azotemia in the dog.

Serum amylase and lipase were measured in 32 healthy dogs and in 55 dogs with serum urea nitrogen values greater than 40 mg/dl, using a nephelometric analyzer (Coleman analyzer model 91). Mean serum amylase and lipase values in normal dogs were 1,085.48 units and 3.98 units, respectively. In dogs with azotemia, the mean serum amylase and lipase values were 1,822.79 units and 12.57 units, respectively. Serum urea nitrogen values could not be correlated with amylase or lipase values.

Effects of ephedrine on cardiovascular function and oxygen delivery in isoflurane-anesthetized dogs.

The hemodynamic effects of 2 dosages of ephedrine were studied in 6 dogs anesthetized with isoflurane only (end-tidal concentration equivalent to 1.5 times minimum alveolar concentration). Following instrumentation, baseline (time 0) measurements included heart rate (HR), respiratory rate, mean arterial blood pressure (MAP), cardiac output, and blood gas tensions. Cardiac index (CI), stroke volume (SV), systemic vascular resistance (SVR), arterial oxygen content (CaO2), and oxygen delivery and consumption (DO2 and VO2, respectively) were calculated. Three dogs were given ephedrine IV at a dosage of 0.1 mg/kg of body weight, and 3 dogs were given ephedrine IV at a dosage of 0.25 mg/kg. Measurements were recorded at 5, 10, 15, 30, and 60 minutes. Each dog then received the alternate dosage of ephedrine, and measurements were again recorded at the same intervals. Effects of ephedrine varied with dosage. Neither dosage was associated with significant changes in pH, PaO2, PaCO2, VO2, or respiratory rate. Ephedrine at a dosage of 0.1 mg/kg caused transient significant increases in MAP, CI, SV, CaO2, and DO2, significant decreases in HR and SVR, and a late, slight decrease in CaO2. Ephedrine at a dosage of 0.25 mg/kg caused a greater and more prolonged increase in MAP, as well as increases in CI, SV, and SVR, and a decrease in HR. The higher dosage of ephedrine also caused a pronounced increase in hemoglobin concentration and CaO2, resulting in a 20 to 35% increase in DO2 throughout the 60-minute experiment.

Cardiovascular effects of xylazine and detomidine in horses.

The cardiovascular effects of xylazine and detomidine in horses were studied. Six horses were given each of the following 5 treatments, at 1-week intervals: xylazine, 1.1 mg/kg, IV; xylazine, 2.2 mg/kg, IM; detomidine, 0.01 mg/kg, IV; detomidine, 0.02 mg/kg, IV; and detomidine, 0.04 mg/kg, IM. All treatments resulted in significantly decreased heart rate, increased incidence of atrioventricular block, and decreased cardiac output and cardiac index; cardiac output and cardiac index were lowest following IV administration of 0.02 mg of detomidine/kg. Mean arterial pressure was significantly reduced for various periods with all treatments; however, IV administration of 0.02 mg of detomidine/kg caused hypertension initially. Systemic vascular resistance was increased by all treatments. Indices of ventricular contractility and relaxation, +dP/dt and -dP/dt, were significantly depressed by all treatments. Significant changes were not detected in stroke volume or ejection fraction. The PCV was significantly reduced by all treatments. Respiratory rate was significantly decreased with all treatments, but arterial carbon dioxide tension did not change. Arterial oxygen tension was significantly decreased briefly with the 3 IV treatments only.

Effect of intra-articular injection of orgotein and saline solution on equine synovia.

Orgotein was injected into the right intercarpal joint of each of 8 horses; the corresponding left joint was left alone (not injected) or was given an injection of normal saline solution. Injection with orgotein caused a transient, marked inflammatory response, evidenced by clinical signs and by increased leukocytes and total protein in the synovia (synovial fluid). Leukocyte numbers and total protein concentration were increased (P less than 0.010) in the orgotein-injected joints within 24 hours. However, saline solution alone also elicited a marked inflammatory response, manifested by increased leukocyte count and total protein concentration. By 7 days, leukocyte and total protein values had returned to approximately base line in all joints.

Cardiopulmonary effects of position in conscious cattle.

The cardiopulmonary effects of 4 positions (standing, right lateral, left lateral, and dorsal recumbency) were evaluated in conscious cattle in which no sedatives or anesthetic drugs were given. Each position was maintained for 30 minutes, during which time there were no significant changes in heart rate, respiratory rate, mean arterial blood pressure, arterial pH, PaCO2, arterial base excess, or venous blood gas values. Significant decreases in PaO2 developed when cattle were in lateral positions and dorsal recumbency. Cardiac index was unchanged in all positions, except in dorsal recumbency at 30 minutes, when it was significantly decreased.

Hemodynamic effects of carbon dioxide during intermittent positive-pressure ventilation in horses.

The hemodynamic effects of high arterial carbon dioxide pressure (PaCO2) during anesthesia in horses were studied. Eight horses were anesthetized with xylazine, guaifenesin, and thiamylal, and were maintained with halothane in oxygen (end-tidal halothane concentration = 1.15%). Baseline data were collected while the horses were breathing spontaneously; then the horses were subjected to intermittent positive-pressure ventilation, and data were collected during normocapnia (PaCO2, 35 to 45 mm of Hg), moderate hypercapnia (PaCO2, 60 to 70 mm of Hg), and severe hypercapnia (PaCO2, 75 to 85 mm of Hg). Hypercapnia was induced by adding carbon dioxide to the inspired gas mixture. Moderate and severe hypercapnia were associated with significant (P less than 0.05) increases in aortic blood pressure, left ventricular systolic pressure, cardiac output, stroke volume, maximal rate of increase and decrease in left ventricular pressure (positive and negative dP/dtmax, respectively), and median arterial blood flow, and decreased time constant for ventricular relaxation. These hemodynamic changes were accompanied by increased plasma epinephrine and norepinephrine concentrations. Administration of the beta-blocking drug, propranolol hydrochloride, markedly depressed the response to hypercapnia. This study confirmed that in horses, hypercapnia is associated with augmentation of cardiovascular function.

Evaluation of creatine kinase and lactate dehydrogenase activities in clinically normal and abnormal equine joints.

Creatine kinase (CK) and lactate dehydrogenase (LD) enzyme activities and isoenzymes were determined for synovial fluid, synovial membrane, and articular cartilage from 24 clinically normal equine tarsocrural (tibiotarsal) and femoropatellar joints. All 3 tissues contained LD isoenzymes LD1 to LD5, and CK isoenzymes BB and MM. The CK isoenzyme MB was not found. The similarities in isoenzyme composition of these 3 tissues made differentiation of the source of LD and CK impossible by isoenzyme pattern alone. Reference values for the total enzyme activities of specific joint tissues also had wide variations. The wide variation in activities, as determined by the enzymatic analysis of synovial fluid and a lack of tissue specificity in clinically normal equine joint tissue, indicated that those values were not predictive for the extent and type of tissue damage in equine joint disease. This hypothesis was confirmed when synovial fluids from 22 abnormal joints were analyzed for LD isoenzymes and total enzyme activity. The various causes of the joint problems were not distinguishable.

Neurodevelopmental outcome in ECMO vs near-miss ECMO patients at 5 years of age.

The objective of this study was to compare the outcome of children at 5 years of age who were treated with extracorporeal membrane oxygenation (ECMO) and those who were critically ill but did not meet ECMO criteria, identified as near-miss ECMO. In one of the longest studies of its kind, we compared the neurodevelopmental outcome of 76 5-year-old ECMO-treated children with 20 5-year-old near-miss ECMO patients with similar primary diagnoses. The two groups were compared for demographic data, level of ventilatory support, and degree of hyperventilation. The comprehensive assessment protocol included an assessment of intelligence (IQ), attainment of preacademic and early academic skills, and parents' report of adaptive behavior. Both groups had similar demographic data and primary diagnosis. The near-miss ECMO patients required increased ventilatory support but not significantly more than the ECMO patients. The cognitive outcome was similar in both groups with mean estimated Full-Scale IQ in the normal range for near-miss and ECMO groups (89 and 97, respectively). Rates of severe mental handicap (FSIQ < 70) (near-miss = 11%, ECMO = 12%) and risk for school failure (near-miss = 38%, ECMO = 37%) were also similar. More parents of near-miss ECMO patients reported immature adaptive skills than did parents of ECMO patients, although the numbers were small in each group. Rates of parent-reported child behavior problems were similar in both groups. ECMO and near-miss ECMO patients have similar cognitive and adaptive outcomes at 5 years of age. A significant number in each group are at risk of school failure and should be closely followed up.

Effects of diazepam and flumazenil on minimum alveolar concentrations for dogs anesthetized with isoflurane or a combination of isoflurane and fentanyl.

OBJECTIVE: To determine the effect of a constant-rate infusion of fentanyl on minimum alveolar concentration (MAC) of isoflurane and to determine the interaction between fentanyl and a benzodiazepine agonist (diazepam) and antagonist (flumazenil) in isoflurane-anesthetized dogs. ANIMALS: 8 mixed-breed adult dogs. PROCEDURE: Dogs were anesthetized with isoflurane 3 times during a 6-week period. After a 30-minute equilibration period, each MAC determination was performed in triplicate, using standard techniques. Fentanyl was administered as a bolus (10 microg/kg of body weight, IV) that was followed by a constant infusion (0.3 microg/kg per min, IV) throughout the remainder of the experiment. After determining isoflurane-fentanyl MAC in triplicate, each dog received saline (0.9% NaCl) solution, diazepam, or flumazenil. After 30 minutes, MAC was determined again. RESULTS: Fentanyl significantly decreased isoflurane MAC (corrected to a barometric pressure of 760 mm Hg) from 1.80+/-0.21 to 0.85+/-0.14%, a reduction of 53%. Isoflurane-fentanyl-diazepam MAC (0.48+/-0.29%) was significantly less than isoflurane-fentanyl-saline MAC (0.79+/-0.21%). Percentage reduction in isoflurane MAC was significantly greater for fentanyl-diazepam (74%), compared with fentanyl-saline (54%) or fentanyl-flumazenil (61%). Mean fentanyl concentrations for the entire experiment were increased over time and were higher in the diazepam group than the saline or flumazenil groups. CONCLUSIONS AND CLINICAL RELEVANCE: Fentanyl markedly decreased isoflurane MAC in dogs. Diazepam, but not flumazenil, further decreased isoflurane-fentanyl MAC. Our results indicate that diazepam enhances, whereas flumazenil does not affect, opioid-induced CNS depression and, possibly, analgesia in dogs.

Cardiopulmonary measurements in dogs undergoing gastropexy without gastrectomy for correction of gastric dilatation-volvulus.

OBJECTIVE: To measure cardiopulmonary variables, including cardiac index, in dogs with naturally acquired gastric dilatation-volvulus (GDV). DESIGN: Prospective clinical study. ANIMALS: 6 dogs with GDV. PROCEDURE: In addition to typical medical and surgical management of GDV, the dorsal metatarsal and pulmonary arteries and right atrium of the dogs were catheterized to obtain cardiopulmonary measurements before and during anesthesia and surgery. RESULTS: All dogs underwent gastropexy but none required gastrectomy. Mean cardiac index and mean arterial blood pressure for this small population of dogs with GDV were not significantly different from those reported for clinically normal awake or anesthetized dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Dogs with naturally acquired GDV without gastric necrosis may not have the classic characteristics, including decreased cardiac index and hypotension, of hypovolemic circulatory shock.