Effects of 5-hydroxytryptophan and m-hydroxybenzylhydrazine associated to Lactobacillus spp. on the humoral response of broilers challenged with Salmonella Enteritidis.

This study investigates the effects of different doses of serotonin, its precursor 5-hydroxytry-ptophan (5HTP), and m-hydroxybenzylhydrazine inhibitor (NSD1015), administered via intraperitoneal for 5 consecutive days, on behavior and average body weight of broilers. We also measured the humoral immune response and quantification of Salmonella Enteritidis in broilers chickens that received the drugs evaluated and a Lactobacillus pool. The study was divided into 3 experiments: Experiment 1--administration of pharmaceuticals with choice of dosage; Experiment 2--administration of pharmaceuticals and a Lactobacillus pool in birds that were not challenged with S. Enteritidis, and Experiment 3--administration of pharmaceuticals and a Lactobacillus pool in birds challenged with S. Enteritidis. The ELISA was used to scan dosages of intestinal IgA and serum IgY. We used colony-forming units to quantify S. Enteritidis. The concentrations of IgA and IgY did not show significant differences (P>0.05) in Experiment 2. In Experiment 3, NSD1015 associated with Lactobacillus determined higher IgA concentrations, promoting greater stimulus to the immune system than 5HTP. Regarding quantification of S. Enteritidis in the cecal content of birds, 5HTP associated to Lactobacillus determined the smallest number of bacteria, showing possible interaction of 5-hydroxytryptophan and Lactobacillus spp. with the immune system of broiler chickens.

Immune response of broiler chickens immunized orally with the recombinant proteins flagellin and the subunit B of cholera toxin associated with Lactobacillus spp.

This study investigated the immune response of broiler chickens with oral treatment of a Lactobacillus spp. pool (PL) associated with microencapsulated recombinant proteins flagellin (FliC) and the subunit B of cholera toxin (CTB). Immune responses were evaluated by measuring IgA from intestinal fluid, serum IgY, and immunostaining of CD8(+) T lymphocytes present in the cecum. The evaluations were performed on d 0, 7, 14, 21, and 28 posttreatment. A significant increase (P < 0.05) was observed in IgA levels in all immunized groups, especially 3 wk after immunization. Treatments 2 (recombinant CTB) and 3 (recombinant FliC+CTB) showed the highest concentrations. Similarly, serum concentrations IgY (µg/mL) increased along the experiment, and the means for treatments 2 and 3 showed significant differences (P < 0.05) compared with controls, reaching concentrations of 533 and 540 µg/mL, respectively. The number of CD8(+) T lymphocytes in all treatments greatly differed (P < 0.05) compared with the negative control at 21 d posttreatment. However, only treatment 2 (recombinant CTB), 4 (PL), and 5 (recombinant FliC+ recombinant CTB + PL) remained significantly (P < 0.05) different from the control at 28 d posttreatment. Thus, it is concluded that the microencapsulated recombinant proteins administered orally to broiler chickens are capable of stimulating humoral and cellular immune response, and the combinations of these antigens with Lactobacillus spp. can influence the population of CD8(+) T cells residing in the cecum.

The effect of the solute on the structure, selected mechanical properties, and biocompatibility of Ti-Zr system alloys for dental applications.

New titanium alloys have been developed with the aim of utilizing materials with better properties for application as biomaterials, and Ti-Zr system alloys are among the more promising of these. In this paper, the influence of zirconium concentrations on the structure, microstructure, and selected mechanical properties of Ti-Zr alloys is analyzed. After melting and swaging, the samples were characterized through chemical analysis, density measurements, X-ray diffraction, optical microscopy, Vickers microhardness, and elasticity modulus. In-vitro cytotoxicity tests were performed on cultured osteogenic cells. The results showed the formation essentially of the α' phase (with hcp structure) and microhardness values greater than cp-Ti. The elasticity modulus of the alloys was sensitive to the zirconium concentrations while remaining within the range of values of conventional titanium alloys. The alloys presented no cytotoxic effects on osteoblastic cells in the studied conditions.

Evaluation of in vitro and in vivo adhesion and immunomodulatory effect of Lactobacillus species strains isolated from chickens.

This study aimed to characterize the in vitro and in vivo adhesion and immunomodulatory effect of Lactobacillus strains isolated from chickens. Lactobacillus samples isolated from 65-wk-old birds were identified by PCR; their adhesion was evaluated in vitro via basement membrane-type cell matrix and in vivo through carboxyfluorescein succinimidyl amino ester staining inoculation in 1-d-old birds and duodenum, jejunum, ileum, and cecum collections at 1, 4, 12, and 24 h after inoculation. The 5 best adhesive samples at the in vitro test formed a pool for total IgA and IgG measurement in sera and intestinal fluid. The birds were divided into groups by inoculation scheme: group 1 was treated with a pool of Lactobacillus spp. at 2-d-old and challenged 1 d later with Salmonella Enteritidis and then treated again with a pool of Lactobacillus spp. at 4 d of age; group 2 was treated with a pool of Lactobacillus spp. at 2 and 4 d of age; group 3 was challenged with Salmonella Enteritidis at 3 d of age; and group 4 was a negative control. Collections were taken at 7, 14, 21, 28, and 35 d after the first inoculation. The results suggest that basement membrane matrix use represents an important technique for triage of samples for subsequent in vivo evaluation and that carboxyfluorescein succinimidyl amino ester staining is efficient for identifying this bacterial characteristic. The Lactobacillus-treated groups (1 and 2) presented the highest IgA concentrations at the end of the experiment (12,054.6 and 10,568.4 ng/mL, respectively). The group 2 IgG values in intestinal fluid exceeded those of the other 3 groups (P < 0.05), peaking at 6.419 ng/mL. In most serum collections, the Lactobacillus-treated groups (1 and 2) did not differ significantly in IgG concentrations (P > 0.05), whereas group 3 presented the highest concentration of this antibody. It is concluded that there was greater adhesion of strains in the cecum and an important correlation between in vitro and in vivo results. These results also suggest the immunomodulatory action of Lactobacillus spp. in the chicken.

Expression and induction of a large set of drug-metabolizing enzymes by the highly differentiated human hepatoma cell line BC2.

The BC2 cell line derived from the human hepatocarcinoma, HGB, undergoes a spontaneous sharp differentiation process in culture as it becomes confluent, remains stably differentiated for several weeks, and may return to proliferation thereafter under appropriate density conditions. The relevance of the line as an hepatic model has been evaluated. Cells synthesize a large number of plasma proteins, and rates of glycogen and urea synthesis increase with time of confluency and become sensitive to insulin, reflecting the process of differentiation. Differentiated BC2 cells express the most relevant cytochrome P-450 (CYP) isozyme activities (CYP1A1/2, 2A6, 2B6, 2C9, 2E1, and 3A4) and conjugating enzymes (glutathione S-transferase and UDP-glucuronyltransferase) and also respond to model inducers. Methylcholanthrene induced an increase in CYP1A1/2 enzyme activity (eightfold), phenobarbital induced CYP2B6 activity (1.7-fold), and dexamethasone induced CYP3A4 activity (fivefold). In parallel, expression of the most relevant liver-enriched transcription factors, HNF-4, HNF-1, C/EBP-alpha and C/EBP-beta mRNAs, was significantly increased in differentiated cultures. This increase was largest in HNF-1 and HNF-4, which supports the idea that a redifferentiation process towards the hepatic phenotype takes place. BC2 is an hepatic cell line that is able to express most hepatic functions, especially the drug-biotransformation function, far more efficiently than any previously described human hepatoma cell line.

New non-woven polyurethane-based biomaterials for the cultivation of hepatocytes: expression of differentiated functions.

A new non-woven polyetherurethane support suitable to host cultured hepatocytes has been developed. Prior to its use in bioreactors and artificial liver devices, the biocompatibility of this new material was investigated. The experiments have shown that the survival and functionality of hepatocytes entrapped in the non-woven polymer were longer than that of monolayer cultured hepatocytes, under serum-free culture conditions. Hepatic specific metabolic functions, namely, synthesis of urea and synthesis and secretion of plasma proteins, were well maintained by hepatocytes entrapped in non-woven polyetherurethane sheets. Cells also retained the expression of biotransformation activities of 7-ethoxycoumarin-O-deethylase as well as CYP2A1, CYP2B1 and CYP3A1. The results presented in this paper point to non-woven polyetherurethane sheets as a suitable biocompatible support for functional, three-dimensional hepatocyte cultures.

Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix.

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).

Adenovirus-mediated gene transfer into human hepatocytes: analysis of the biochemical functionality of transduced cells.

The use of replication-defective adenoviruses to deliver transgenes into hepatocytes seems to be a promising approach to human liver gene therapy. However, the effects that the adenovirus-mediated expression of a foreign gene could have on the expression of other hepatic characteristic genes have not yet been properly examined. We have investigated this problem by using human hepatocytes infected with a recombinant E-1 defective adenovirus that carried a modified lacZ gene. The analysis of the biochemical functionality of transduced cells showed that the use of adenovirus: (1) was a very efficient way to introduce a foreign gene into human hepatocytes (80% transduced cells after 1 h contact, at an MOI of 15; approximately 100% transduced cells at an MOI of 20); (2) allowed the expression of the transgene to levels that enabled cells effectively to use lactose as an energy source; (3) does not affect urea synthesis, plasma protein synthesis and xenobiotic biotransformation activities (1A2, 2A1, 2B6, 3A3/5). Glycolysis was moderately increased (approximately 20%), while gluconeogenesis decreased (approximately 20%) in transduced hepatocyte; moreover, (4) the expression of inducible genes (acute-phase plasma proteins, CYPs) was not impaired in transduced human hepatocytes upon stimulation with IL-6 or methylcholantrene. The results of this research support the idea that efficient expression of transgenes can be achieved in human hepatocytes by means of adenoviral transduction, without altering these characteristic hepatic biochemical functions.

The influence of acute and chronic alcohol treatment on brain edema, cerebral infarct volume and neurological outcome following experimental head trauma in rats.

The aim of this study was to determine the influence of acute and chronic ethanol treatment on neurological outcome following head trauma in rats. Eight-two Sprague-Dawley rats were divided into 10 groups. Four groups received sham head trauma (surgical incision of the scalp but no trauma) and were treated with (A) nothing, (B) chronic ethanol (6% ethanol in drinking water for 40 days), (C) acute ethanol 1.5 g/kg, intraperitoneally (IP) and (D) acute ethanol 3 g/kg IP. Four groups (E-H) received the same treatment; in addition, head trauma was delivered using a weight-drop device to the left cranium (2 h after ethanol treatment in the acute ethanol groups). To assess the influence of acute plus chronic alcohol and whether the glutamate antagonist ketamine can modify the neurologic outcome following head trauma, two additional head trauma groups were studied: group I received both acute and chronic ethanol treatment and group J received acute ethanol (1.5 g/kg) IP plus ketamine (180 mg/kg). Neurologic assessment was performed at 1, 2, 4, 8, 10, and 24 hours after head trauma or sham trauma in all animals who survived the treatment (omitted in group J animals while still under the anesthetic influence of ketamine). At the end of 24 hours, or at the time of death, the animals were decapitated. Specific gravity was determined from brain tissues adjacent to the injury and the contralateral hemispheres and the volume of hemorrhagic necrosis was quantified. None of the rats in the sham trauma groups died or had neurologic deficits. Of the head trauma groups, the mortality in animals that received 3 g/kg of ethanol and the animals that received acute plus chronic ethanol treatment was 100% before the end of 24 hours. Fifty percent of the animals receiving low-dose acute ethanol treatment (1.5 g/kg) died before 24 hours. In contrast, the mortality in the other groups was only 30% (p < 0.05). The neurologic severity score was significantly higher (greater neurological deficit) in the surviving animals that received acute ethanol treatment at 10 and 24 hours than in rats receiving no ethanol or chronic ethanol treatment. Specific gravity was also lower in the acute ethanol-treated groups compared with no ethanol, chronic ethanol, and acute ethanol plus ketamine groups (p < 0.03). Based on these observations, we conclude that in this rat head trauma model acute ethanol treatment increases mortality, neurological deficit, hemorrhagic necrosis volume, and brain edema. On the other hand, chronic ethanol treatment has no apparent effect and ketamine treatment does not counteract the effect of acute ethanol treatment.

Effect of mannitol on cerebrospinal fluid dynamics and brain tissue edema.

Mannitol is used widely to decrease intracranial pressure (ICP); however, the mechanism by which this effect occurs is unclear. This study was designed to examine the effects of mannitol on cerebrospinal fluid (CSF) formation rate (Vf), resistance to reabsorption of CSF (Ra), and brain tissue water content (BTWC). Eighteen New Zealand White rabbits were allocated into one of three groups and studied at baseline and at two sequential doses of 20% mannitol: 0.75 g/kg followed by 4.4 mg.kg-1.min-1, and 2.0 g/kg (1.25 g/kg added to the initial dose of 0.75 g/kg) followed by 8.6 mg.kg-1.min-1. In Group 1, closed ventriculocisternal perfusion (VCP) was performed to determine changes in ICP due to mannitol. In Group 2, the increase in CSF osmolality due to mannitol was determined. In Group 3, mock CSF was used for open VCP to determine Vf and Ra. At the conclusion of each study, brain tissue samples were taken for determination of BTWC. Mannitol increased CSF and plasma osmolality. Ra was increased by 104% with the low dose of mannitol and not significantly changed by the high dose. Mannitol decreased BTWC, Vf (by 49% with the high dose), ICP, and hematocrit. The authors conclude that two of the mechanisms contributing to decreased ICP with mannitol are: 1) decreased CSF volume as indicated by decreased Vf, and 2) decreased brain tissue volume as indicated by decreased BTWC.

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo.

High yields of human hepatocytes (up to 23 X 10(6) viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham's F12 containing 0.2% bovine serum albumin, 10(-8) M insulin, and 10(-8) M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250 +/- 177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50 +/- 0.17 nmol glucose.mg-1.min-1) similar to that reported for human liver. Insulin at 10(-8) M activated glycolysis (X1.40) and glycogenesis (X1.34), and glucagon at 10(-9) M stimulated gluconeogenesis (X1.35) and glycogenolysis (X2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein, haptoglobin, alpha 2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10(-9) M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol.mg-1 cell protein.min-1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol.mg-1). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.

The potential use of cultured hepatocytes in predicting the hepatotoxicity of xenobiotics.

1. A protocol is proposed for screening for hepatotoxicity of xenobiotics in vitro in which hepatocytes exposed to the compounds are evaluated for both cytotoxic and metabolic effects. Four established hepatotoxins have been studied. 2. alpha-Amanitin at 1.5 pg/mg cell protein inhibited RNA synthesis by 93% and reduced albumin synthesis to 56% of the control after 13 h treatment. 3. D-Galactosamine at 40 microM inhibited glycogen synthesis by 31%, glucuronidation of p-nitrophenol by 13% and albumin synthesis by 10%, and produced an increase in cytosolic enzyme leakage. 4. Thioacetamide decreased ureogenesis after 24 h of treatment at 230 microM (31% inhibition) and after 48 h at 2.3 microM (25% inhibition). 5. Ultrastructural alterations of hepatocytes were found after 48 h exposure to 1 mM acetaminophen and were preceded by extensive leakage of the enzymes GOT and LDH. Membrane damage was observed after 24 h exposure to 0.1 mM acetaminophen.