Porcine hepatocyte-Kupffer cell co-culture as an in vitro model for testing the efficacy of anti-inflammatory substances.

As Kupffer cells are highly involved in the regulation of hepatic inflammatory response, the main goal of this study was to improve and to characterize a hepatocyte-Kupffer cell co-culture of pig origin for modelling endotoxin-induced hepatic inflammation and for testing the efficacy of potential anti-inflammatory substances. This monolayer co-culture was prepared from primary isolated swine hepatocytes and Kupffer cells in the ratio of 6:1 and 2:1, mimicking different states of liver inflammation. The prepared cell cultures were characterized by immunohistochemical CD-68 detection. Lipopolysaccharide (LPS) challenge of both co-cultures resulted in elevated interleukin-8 (IL-8) and that of 6:1 co-cultures in increased IL-6 production with a higher extent than on hepatocyte monocultures, justifying the key role of Kupffer cells in pro-inflammatory cytokine production. LPS-induced IL-8 production was successfully attenuated by concomitant application of both sodium butyrate and terpinen-4-ol on hepatocyte monocultures, but not on co-cultures, demonstrating the importance of the presence of Kupffer cells in cell cultures as inflammatory models. Based on these initial data, the applied porcine primary hepatocyte-Kupffer cell co-culture is suggested to be a proper tool for in vitro investigations on liver physiology and hepatic inflammation in pigs and can be used as a useful model mimicking in vivo conditions in veterinary research.

Feed-drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens.

The expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition-derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed-drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP-inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non-specific CYP-inducer, simultaneously with two different doses of intra-ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB-triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP-inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP-inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition.

Effects of oral butyrate application on insulin signaling in various tissues of chickens.

The influence of butyrate on insulin signaling in chickens was studied because butyrate is produced during microbial fermentation in the large intestine of birds, and butyrate is widely used as a feed additive in animal production. Ross 308 broiler chickens received a daily intraingluvial bolus of sodium butyrate (0.25 g/kg body weight) on days 20-24 of life (n = 10). Plasma butyrate concentration increased after receiving oral butyrate treatment (P < 0.001). Oral butyrate application was associated with decreased protein expression of insulin receptor ß subunit (IRß) in liver (P = 0.008) and both abdominal (P = 0.003) and subcutaneous adipose tissue (P < 0.001), but with elevated IRß expression in muscle (P = 0.045), assessed by Western blotting. The quantity of hepatic phosphatidyl-inositol-3-kinase was reduced in the butyrate-treated group (P = 0.007); further, mammalian target of rapamycin was downregulated by butyrate in liver (P < 0.001) and subcutaneous adipose tissue (P = 0.038). Oral butyrate application provoked reduced systemic insulin sensitivity in chickens, indicated by elevated fasting blood glucose and subsequently, insulin level. However, responses of insulin signaling cascade to butyrate were tissue specific, suggesting that butyrate could act on glucose shifting among tissues by selectively increasing the glucose uptake of skeletal muscle via IRß upregulation.

Effects of Lactobacillus plantarum 2142 and sodium n-butyrate in lipopolysaccharide-triggered inflammation: comparison of a porcine intestinal epithelial cell line and primary hepatocyte monocultures with a porcine enterohepatic co-culture system.

This study was based on our previously developed double-layered enterohepatic co-culture system, composed of nontumorigenic porcine intestinal epithelial cell line (IPEC-J2) and primary culture of porcine hepatocytes. The anti-inflammatory effect of spent culture supernatant of Lactobacillus plantarum 2142 (Lp2142; 13.3%) and sodium n-butyrate (2 mM) was tested on IPEC-J2 and hepatocyte monocultures as well as on the gut-liver co-culture. To mimic inflammation, lipopolysaccharide (LPS; 1 and 10 µg/mL) was applied. Production of IL-8 and IL-6 was measured as a marker of inflammatory responses. The paracellular permeability of the intestinal epithelium was also monitored by fluoresceinisothiocyanate-labeled dextran 4 assay. Significant increase of IL-8 concentration was observed in the IPEC-J2 monoculture (P < 0.01) while the level of IL-6 was not changed following LPS treatment. Concentration of IL-8 and IL-6 was grown significantly in hepatocyte monocultures (P < 0.05 and P < 0.001) as well as in the co-culture after 10 µg/mL LPS treatment (P < 0.001 and P < 0.001). One microgram per milliliter LPS caused elevated IL-8 level in the co-culture (P < 0.001) and in the hepatocyte monoculture (P < 0.01), while it caused increased IL-6 level only in the hepatocytes (P < 0.001). Production of IL-8 was significantly decreased by butyrate in case of 1 µg/mL as well as 10 µg/mL LPS exposure in the co-culture (P < 0.001). Application of butyrate also reduced IL-6 level in the co-culture after 10 µg/mL LPS treatment (P < 0.01). Lactobacillus plantarum 2142 decreased IL-8 level after incubation with 1 µg/mL LPS (P < 0.001), while in case of 10 µg/mL LPS treatment only a marginal lowering in IL-8 (P = 0.064) release was measured. The IL-6 concentration was significantly reduced (P < 0.01 in case of 1 µg/mL LPS treatment) by Lp2142 in the co-culture. Contrarily, the elevated IL-8 and IL-6 level of hepatocytes has not been reduced in case of either butyrate or Lp2142 addition. The enterohepatic co-culture model offers a possibility for fast and reliable screening of new candidates against enteric inflammation, which are of special interest in porcine medicine and health management. According to our results, Lp2142 and butyrate both seem to be effective as anti-inflammatory agents in LPS-triggered inflammatory response, tested in the gut-liver co-culture model.

Effects of dietary sodium butyrate on hepatic biotransformation and pharmacokinetics of erythromycin in chickens.

Butyrate, a commonly applied feed additive in poultry nutrition, can modify the expression of certain genes, including those encoding cytochrome P450 (CYP) enzymes. In comparative in vitro and in vivo experiments, the effect of butyrate on hepatic CYP genes was examined in primary cultures of chicken hepatocytes and in liver samples of chickens collected from animals that had been given butyrate as a feed additive. Moreover, the effect of butyrate on the biotransformation of erythromycin, a marker substance for the activity of enzymes of the CYP3A family, was investigated in vitro and in vivo. Butyrate increased the expression of the avian-specific CYP2H1 both in vitro and in vivo. In contrast, the avian CYP3A37 expression was decreased in hepatocytes following butyrate exposure, but not in the in vivo model. CYP1A was suppressed by butyrate in the in vitro experiments, and overexpressed in vivo in butyrate-fed animals. The concomitant incubation of hepatocytes with butyrate and erythromycin led to an increased CYP2H1 expression and a less pronounced inhibition of CYP3A37. In in vivo pharmacokinetic experiments, butyrate-fed animals given a single i.m. injection of erythromycin, a slower absorption phase (longer T(half-abs) and delayed T(max)) but a rapid elimination phase of this marker substrate was observed. Although these measurable differences were detected in the pharmacokinetics of erythromycin, it is unlikely that a concomitant application of sodium butyrate with erythromycin or other CYP substrates will cause clinically significant feed-drug interaction in chickens.

Satisfaction survey of Greek inpatients with brain cancer.

PURPOSE: To investigate brain cancer patients' satisfaction hospitalised in a tertiary care university public hospital in Alexandroupolis, Greece, in order to improve medical, nursing, and organizational-administrative services. METHODS: This cross-sectional study involved 163 patients having been hospitalised for at least 24 hours. The patients were asked to fill in a satisfaction questionnaire previously approved by the Greek Ministry of Health. Four aspects of satisfaction were investigated (medical, hotel accommodation/ organisational facilities, nursing, global). Using Principal Component Analysis, summated scales were formed and tested for internal consistency using Cronbach's alpha coefficient. The non parametric Spearman's rank correlation coefficient was also used and the threshold p-value for statistical significance (2-sided) was set at 0.05. RESULTS: The results revealed a high degree of global satisfaction (73.31%), yet satisfaction was higher for the medical (88.88%) and nursing (84.26%) services. Moreover, satisfaction derived from the accommodation facilities and the general organisation was found to be more limited (74.17%). Statistically significant differences (based on various demographic variables) in the participants' global satisfaction were not observed. On the contrary, self-assessment of health status at admission was negatively correlated with medical (r(s)=-0.157, p=0.045) and nursing (r(s)=-0.168, p=0.032) satisfaction. Greek citizenship contributed to bigger satisfaction scores in the accommodation/organisational facilities dimension (r(s)=0.158, p=0.044). Finally, age was positively linked to nursing satisfaction (r(s)=0.181, p=0.02). CONCLUSION: The present study confirmed in part the results of previously published Greek surveys assessing general patient populations. However, more studies are urgently needed to confirm these findings in a much bigger brain cancer population.

Glasgow coma scale and APACHE II system data--are they normally distributed?

OBJECTIVE: To verify whether the Glasgow Coma Scale (GCS) and APACHE II system scores are normally distributed. METHOD: Medical records of seventy four head injured patients were reviewed. GCS and APACHE II data were compared to a standard normal distribution. The chi2 goodness of fit test, the one sample Kolmogorov-Smirnov test, the Lilliefors test, the Shapiro-Wilk test, and calculation of skewness and kurtosis were performed. Frequency histograms and normal probability plots were also constructed. RESULTS: GCS data were not found to be normally distributed with a Kolmogorov-Smirnov statistic of 3.181 (p=0.000). In contrast, APACHE II data presented a normal distribution with a Kolmogorov-Smirnov statistic of 0.704 (p=0.704). CONCLUSIONS: We demonstrated that GCS data do not follow the normal distribution, and thus nonparametric tests should be employed when dealing with such data. Furthermore, APACHE II data follow the normal distribution, and parametric tests could be considered, even though nonparametric tests are still preferred.

Intraperitoneal cerebrospinal fluid pseudocyst. A rare complication of ventriculoperitoneal shunt.

The abdominal intraperitoneal cerebrospinal fluid pseudocyst is a rare but important complication in patients with ventriculoperitoneal shunts. We report a case of a 31-year-old female, in which a large abdominal pseudocyst was developed 1 year after insertion of a ventriculoperitoneal shunt for hydrocephalus. The abdominal CT scan and the ultrasonographical evaluation of the abdomen showed a well defined, cystic mass lesion with a volume of 50 cm3, in the recessus hepato-renal. The peritoneal tip of the shunt was located within the mass lesion. A distal externalization of the peritoneal catheter without excision of the pseudocyst was performed. Cerebrospinal fluid culture demonstrated a Staphylococcus epidermis infection and adequate antibiotic treatment was administrated. The previous symptoms improved 4 weeks later and a new catheter was placed intraperitoneally in a different quadrant. The postoperative course was uneventful. We suggest that chronic inflammation or subclinical peritonitis is a predisposing factor for this complication.