Development of an in vitro macrophage screening system on the immunomodulating effects of feed components.

BACKGROUND: While feed components capable of modulating the immune system are highly sought after and marketed, often little evidence is available to support functional immune response claims. Thus, a high-throughput in vitro cell screening system was developed to test these compounds for innate immune signaling effects, using Saccharomyces cerevisiae and its cell wall components in addition to lauric acid and its esters as models in two separate experiments. This screening system utilized RAW 264.7 murine macrophages to assess live S. cerevisiae cells and S. cerevisiae-derived cell wall components ß-glucan, mannan, and zymosan (a crude cell wall preparation containing both ß-glucan and mannan). D-mannose was also evaluated as the monomer of mannan. We also examined the effect of a saturated fatty acid (C12:0, lauric acid) and its esters (methyl laurate and glycerol monolaurate) on innate immune cell activation and cellular metabolism. RAW cells were transfected with a vector that drives expression of alkaline phosphatase upon promoter activation of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB), a major inflammatory/immune transcription factor. RAW cells were incubated with 0.01, 0.1 or 1 mg/mL of yeast compounds alone or RAW cells were challenged with LPS and then incubated with yeast compounds. In a separate experiment, RAW cells were incubated with 0, 0.5, 2.5, 12.5, 62.5, and 312.5 µmol/L of lauric acid, methyl laurate, or glycerol monolaurate alone, or RAW cells were challenged with LPS and then incubated with fatty acid treatments. RESULTS: Treatment with zymosan or ß-glucan alone induced NFκB activation in a dose-dependent manner, whereas treatment with D-mannose, mannan, or live S. cerevisiae cells did not. Post-treatment with mannan after an LPS challenge decreased NFκB activation, suggesting that this treatment may ameliorate LPS-induced inflammation. Slight increases in NFκB activation were found when fatty acid treatments were applied in the absence of LPS, yet substantial reductions in NFκB activation were seen when treatments were applied following an LPS challenge. CONCLUSIONS: Overall, this cell screening system using RAW macrophages was effective, high-throughput, and sensitive to feed components combined with LPS challenges, indicating modulation of innate immune signaling in vitro.

A compact flexible pellet injector for the TJ-II stellarator.

A compact pellet injector is being built for the TJ-II stellarator. It is an upgraded version of the "pellet injector in a suitcase" developed at Oak Ridge National Laboratory and installed on the Madison Symmetric Torus where it continues to be used in many plasma experiments. The design aim is to provide maximum flexibility at minimal cost, while allowing for future upgrades. It is a four-barrel system equipped with a cryogenic refrigerator for in situ hydrogen pellet formation, a combined mechanical punch/propellant valve system, pellet diagnostics, and an injection line, destined for use as an active diagnostic and for fueling. In order to fulfill both objectives it will be sufficiently flexible to permit pellets, with diameters from 0.4 to 1 mm, to be fabricated and accelerated to velocities from 150 to approximately 1000 m s(-1).

Interleukin-5 inhibition of biliary cell chloride currents and bile flow.

Recent studies have detected significant elevations of interleukin (IL)-5 mRNA in the liver parenchyma of patients with both primary biliary cirrhosis and acute rejection after liver transplantation. In both of these disorders, intrahepatic biliary epithelial cells (BECs) are the targets of injury. We hypothesized that BECs may themselves express IL-5 receptors that may modulate key biliary functions. RNAs coding for IL-5alpha and -beta receptors were amplified by RT/PCR from a biliary cell line derived from a human cholangiocarcinoma (Mz-ChA-1) and verified by DNA sequencing. IL-5 receptor distribution was detected immunocytochemically on Mz-ChA-1 cells, immortalized murine BEC, bile duct-ligated rat liver, and isolated cholangiocytes. Patch-clamp studies on Mz-ChA-1 cells showed that IL-5 inhibits 5'-N-ethylcarboxamidoadenosine-stimulated chloride currents. Additional functional studies showed that IL-5 inhibits secretin-induced bile flow. We conclude that BECs express IL-5 receptors and that IL-5 modulates BEC chloride currents and fluid secretion. Since IL-5 has previously been associated with cholestatic liver disease, we speculate that IL-5 may contribute to liver injury through its effects on biliary secretion.

Alternative splice variant of gamma-calmodulin-dependent protein kinase II alters activation by calmodulin.

Calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous, multifunctional enzyme family involved in the regulation of a variety of Ca(2+)-signaling pathways. These family members are expressed from four highly homologous genes (alpha, beta, gamma, and delta) with similar catalytic properties. Additional isoforms of each gene, created by alternative splicing of variable regions I-XI, are differentially expressed in various cell types. gammaB, gammaC, gammaD, gammaE, gammaF, gammaGs, and gammaH CaMKII isoforms are expressed in the biliary epithelium; however, little is known about their roles in these cells. We began our studies into the function of these variable regions by examining the effects of variable region I on kinase activation and calmodulin binding. Activities and calmodulin binding properties of gammaB and gammaGs, which differ only by the exclusion or inclusion of this region, were compared. The K(0.5) for calmodulin was 2.5-fold lower for gammaGs than gammaB. In contrast, gammaB bound calmodulin more tightly in a calmodulin overlay assay. Mutation of variable regions I's charged residue, gammaGs-R318E, resulted in an enzyme with intermediate activation properties but a calmodulin affinity similar to gammaB. Thus, variable region I appears to modulate calmodulin sensitivity, in part, through charge-charge interactions. This altered threshold of activation may modulate cellular responses to gradients of Ca(2+)/calmodulin in the biliary tract.

Cholestatic liver diseases in adults.

Cholestatic liver diseases are a diverse group of disorders that are recognized by either increases in laboratory studies or the appearance of jaundice, fatigue, pruritus, and/or complications of cirrhosis. The etiologies for most forms of these diseases are unknown. In this paper, diagnostic and therapeutic strategies are reviewed for select forms of cholestatic disorders and for the management of shared complications of cholestatic illness.

5-year incidence of adenomas after negative colonoscopy in asymptomatic average-risk persons [see comment].

BACKGROUND & AIMS: Cost-effectiveness of colorectal cancer screening will be maximized by selecting the widest screening intervals that effectively prevent cancer mortality. However, data on the incidence of neoplasia in persons with no abnormal findings on initial examination are limited. The aim of this study was to describe the incidence of colonic neoplasia 5 years after negative screening colonoscopy in asymptomatic average-risk persons. METHODS: We previously reported the results of screening colonoscopy in 496 asymptomatic average-risk persons, 368 of whom had no neoplasia identified. Colonoscopy to the cecum was performed in 154 of these persons at a mean of 66 months after the initial negative colonoscopy. RESULTS: Forty-one (27%) had at least one adenoma, but only 1 person had an adenoma > or = 1 cm and none had cancer, severe dysplasia, or villous or tubulovillous histology. Hyperplastic polyps at the initial examination did not predict incident adenomas. Regular nonsteroidal anti-inflammatory drug use was associated with a decreased rate of incident adenomas. CONCLUSIONS: In average-risk persons, the interval between screening examinations can be safely expanded beyond 5 years, provided the initial examination is a carefully performed complete colonoscopy that is negative for colonic adenomas or cancer.

Survey of cystic fibrosis transmembrane conductance regulator genotypes in primary sclerosing cholangitis.

A variety of cholestatic liver diseases appear to primarily affect the biliary epithelium, including cystic fibrosis (CF). CF results from a defect in the chloride channel protein, cystic fibrosis transmembrane conductance regulator (CFTR). Although the majority of CF patients have a genomic deletion in deltaF508, other mutations of CFTR may result in less severe clinical presentations and outcomes. Recently, CFTR has been shown to be involved in secretin-stimulated choleresis in intrahepatic bile duct epithelial cells. Cholestasis in cystic fibrosis appears to result from defective chloride transport across the biliary epithelium and is the only cholestatic disease of bile ducts for which a cellular defect has been identified. Primary sclerosing cholangitis (PSC) is a cholestatic disease with histological and cholangiographic features similar to CF. The purpose of this pilot study was to explore whether there is an increased prevalence of CFTR mutations. Two patients exhibited mutations in one allele, yielding a carrier rate of 10.6%, not statistically different from the general U.S. population carrier rate of 4%.

Human biliary epithelial cell line Mz-ChA-1 expresses new isoforms of calmodulin-dependent protein kinase II.

BACKGROUND & AIMS: Calmodulin-dependent protein kinase II is a family of closely related multimeric enzymes that regulate a wide variety of cellular processes. In biliary epithelial cells, this kinase seems to regulate Ca(2+)-dependent CI- currents. The aim of this study was to identify isoforms of this kinase expressed in biliary cells. METHODS: Sequencing of reverse-transcription polymerase chain reaction products identified multiple isoforms in Mz-ChA-1 cells. RESULTS: Two previously identified isoforms (gamma B and gamma C) and three new isoforms (gamma D, gamma E, and gamma F) of calmodulin-dependent protein kinase II were identified. Each of the novel isoforms contains a unique insert of 114 base pairs in the association region. This insert lies outside the previously identified variable region. In addition, gamma D and gamma F contained other deletions (42 and 69 base pairs, respectively) in the variable region. These isoforms are expressed in a variety of tissues, including biliary epithelial and gallbladder cells, but only gamma C is expressed in rat hepatocytes. CONCLUSIONS: Identification of these biliary kinase isoforms paves the way for future studies that will elucidate the role of individual isozymes in agonist-stimulated biliary Cl- and fluid secretion.

Ursodeoxycholate increases cytosolic calcium concentration and activates Cl- currents in a biliary cell line.

BACKGROUND & AIMS: Ursodeoxycholate (UDC) stimulates a bicarbonate-rich choleresis, but the cellular mechanisms involved are not fully established. Because ductular secretion also increases biliary HCO3-concentration, the purpose of this study was to evaluate whether UDC has direct effects on duct cells by measuring intracellular calcium concentration ([Ca2+]i) and membrane Cl- permeability in Mz-ChA-1 human cholangiocarcinoma cells. METHODS: Intracellular calcium levels were measured using fura-2 fluorescence. Membrane Cl- permeability was assessed in subconfluent monolayers using 125I efflux and in individuals cells using whole-cell patch clamp techniques. RESULTS: Exposure to UDC (2.5 mmol/L) increased [Ca2+]i from 180 +/- 25 to 639 +/- 84 nmol/L due to release of Ca2+ from intracellular stores and stimulated 125I efflux approximately threefold above basal levels. Exposure to extracellular (1.25 mmol/L) or intracellular (100 mumol/L) UDC activated currents carried by Cl- ions; intracellular UDC increased current density from 4.7 +/- 1.3 to 32.5 +/- 8.8 pA/pF. UDC-stimulated currents were inhibited by chelation of intracellular calcium. CONCLUSIONS: UDC in pharmacological concentrations increases [Ca2+]i and stimulates Cl- efflux through opening of Cl- channels in biliary cells. We speculate that UDC could increase bile flow by direct stimulation of ductular secretion and may be of therapeutic benefit to patients with cystic fibrosis who have impaired adenosine 3',5'-cyclic monophosphate-dependent biliary secretion.

ATP-activated chloride permeability in biliary epithelial cells is regulated by calmodulin-dependent protein kinase II.

Previous studies in freshly isolated rat biliary epithelial cells and in the human cholangiocarcinoma cell line Mz-ChA-1 have demonstrated that ATP activates a calcium-dependent chloride conductance. The coupling between the rise in intracellular calcium and activation of chloride channels has not previously been investigated. In the present study, we evaluated the potential role of calmodulin-dependent protein kinase II (CaMKII) in ATP-activated chloride permeability in Mz-ChA-1 cells. ATP stimulated [125I] efflux, a marker for Cl- movement. Peak efflux rates were inhibited by approximately 60% in cells pretreated with the calmodulin antagonist, W-7. In whole-cell patch clamp recordings, ATP and ionomycin activated calcium-dependent Cl- currents. Pretreatment of cells with the CaMKII inhibitor KN-62 blocked activation by either agent. It is concluded that calcium-dependent activation of chloride currents in Mz-ChA-1 cells is coupled to a CaMKII-dependent process.

Adenosine triphosphate activates ion permeabilities in biliary epithelial cells.

BACKGROUND/AIMS: The biliary epithelium contributes to bile formation through absorption and secretion of fluid and electrolytes. The effects of extracellular nucleotides on membrane ion transport were assessed in isolated bile duct cells from rats and Mz-ChA-1 cells from a human cholangiocarcinoma. METHODS: The rates of efflux of 125I and 86Rb were used to assess membrane Cl- and K+ permeabilities, respectively. Patch clamp recordings of whole cell currents were used to evaluate the properties of adenosine triphosphate (ATP)-activated currents. RESULTS: Purinergic receptor agonists ATP and uridine triphosphate stimulated 125I and 86Rb efflux about twofold above basal levels. The effects were reproduced by a nonhydrolyzable analogue of ATP (adenosine 5'-O-[3-thiophosphate]) and were unaffected by an adenosine receptor blocker xanthine amine congener. 125I efflux was also stimulated by adenosine and its receptor agonists 5'-N-ethylcarboxamidoadenosine, N6-(2-phenylisopropyl)adenosine; these effects were inhibited by xanthine amine congener, suggesting a separate adenosine receptor. ATP, adenosine 5'-O-(3-thiophosphate), and uridine triphosphate each stimulated release of Ca2+ from intracellular stores, whereas adenosine had no effect. In whole cell recordings of Mz-ChA-1 cells, ATP activated an early transient outward current consistent with a K+ conductance and a later, sustained inward current consistent with a Cl- conductance. CONCLUSIONS: Biliary cells possess at least two classes of nucleotide receptors that modulate membrane ion permeability through Ca(2+)-dependent and -independent pathways, and ATP may be involved in the regulation of biliary secretion.

Secretin activates Cl- channels in bile duct epithelial cells through a cAMP-dependent mechanism.

Using patch-clamp recording techniques, we assessed the effects of secretin on membrane ion channel activity in isolated rat bile duct epithelial cells. In the whole cell configuration, secretin activated an inward membrane current at -40 mV in 6 of 13 cells, and increased current density from 17 +/- 8 to 98 +/- 33 pA/pF. Secretin-stimulated currents reversed near the equilibrium potential for Cl- and exhibited a linear current-voltage relationship. In the cell-attached configuration, secretin activated low-conductance channels in 73% (11 of 15) of patches. Similar channels were activated by forskolin, suggesting that adenosine 3',5'-cyclic monophosphate (cAMP) is involved as a second messenger. At the resting membrane potential, channels carried inward membrane current and had a slope conductance of 10 +/- 1 pS. In excised patches, addition of purified catalytic subunit of cAMP-dependent protein kinase (protein kinase A) to the cytoplasmic surface activated channels in four of six attempts. With equal Cl- concentrations in bath and pipette, channels had a linear slope conductance of 13 +/- 2 pS and currents reversed near 0 mV. Partial substitution of pipette Cl- with gluconate caused a shift in reversal potential in the direction anticipated for a Cl(-)-selective channel (gluconate to Cl- permeability ratio of 0.21 +/- 0.05, n = 4). Thus in bile duct epithelial cells, exposure to secretin activates low-conductance, Cl(-)-selective channels, probably through a cAMP-dependent mechanism. This likely contributes to secretin-dependent choleresis.

Cl- and K+ transport in human biliary cell lines.

BACKGROUND: The cellular mechanisms which contribute to billing secretion and absorption are not fully defined. The purpose of these studies was to evaluate the membrane ion transport properties of Mz-ChA-1 and Sk-ChA-1 cell lines derived from human biliary tumors. METHODS: In cultured cells, 125I and 36Cl efflux rates were used to assess membrane anion permeability, and 86Rb efflux rates were used to assess K+ permeability. RESULTS: Sections of tumors grown on BALB/Urd mice were used for morphological evaluation and for detection of cystic fibrosis transmembrane conductance regulator (CFTR), the protein product of the cystic fibrosis gene. There was organized development of ductular structures and cells stained for gamma-glutamyl transpeptidase and CK-19. Immunoperoxidase staining for CFTR, which is likely a Cl- channel, was also present. Increases in intracellular Ca2+ stimulated by exposure to ionomycin or thapsigargin increased efflux of 125I, 36Cl, and 86Rb. Efflux of 125I was greater than 36Cl, and anion efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Increases in 5'-cyclic adenosine monophosphate increased efflux of 36Cl greater than 125I but had no effect on 86Rb efflux. Both cell lines possess bumetanide-sensitive 86Rb uptake consistent with possible Na+/K+/2Cl- cotransport. CONCLUSIONS: These human cell lines retain certain phenotypic features of differentiated biliary cells and may be useful for further investigation of biliary fluid and electrolyte transport.

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells.

Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n = 10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl- permeability ratio of 0.17 +/- 0.04) and had a unitary conductance of approximately 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5'-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5' guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both Gi alpha-2 and Gi alpha-3 were present in significant amounts, whereas Gi alpha-1 and Go alpha were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.

Characterization of high-conductance anion channels in rat bile duct epithelial cells.

We have utilized patch clamp recording techniques to identify a high-conductance anion channel in the plasma membrane of rat bile duct epithelial cells. Cells were isolated from the intrahepatic bile duct 2-6 wk after bile duct ligation. Channels were present in 27% (28/102) of excised patches, and, with 150 mM Cl- in bath and pipette solutions, the slope conductance of the fully open level was approximately 364 +/- 18 pS (n = 8) with current reversal = 0 +/- 1 mV. Channel characteristics were not affected by substitution of K+ for Na+ in the pipette solution; but substitution of HCO3-, gluconate, or increased NaCl caused a shift in the reversal potential toward the new equilibrium potential for Cl-. The permeability ratios were PHCO3-/PCl- = 0.51 +/- 0.03 (n = 5), Pgluconate/PCl- = 0.12 +/- 0.04 (n = 7), and PNa+/PCl- = 0.11 +/- 0.02 (n = 3). Current transitions to a subconductance level at 72% of the fully open level were present in most studies. Channel open probability was greatest near 0 mV and decreased rapidly outside of -20 to +20 mV because of voltage-dependent channel closure. The time course for current relaxation of summed single channel currents could be described by a single exponential with more rapid channel closure as the magnitude of the voltage step away from 0 mV increased. In the cell-attached configuration, the channel was rarely open (4/35, 11%) but opening could be induced by negative pipette pressure (5/14, 35%). Possible physiological roles for this channel are discussed.

The patterns of brain gangliosides of Rana catesbeiana during metamorphosis and in the adult.

Ganglioside patterns were quantitated for brains of bullfrog (Rana catesbeiana) at three stages of metamorphosis and adult. At each stage nine gangliosides were identified by mobility on silica gel thin-layer chromatograms, and quantitated on the basis of sialic acid content. A single band with chromatographic mobility close to that of GD2, and doublets close to GT1b and GQ were quantitatively the major ones (over 16% each). Doublets close to GM1 and GD1b and a single band slightly behind GD1a made up 5-10% each. A doublet comigrating with GM3, and bands close to GM2 (trace) and GD3 were present in smaller amounts. The only developmental trend was a slight increase in the proportion of the band close to GD3 from 2.4% (early prometamorphic phase) to 9.1% (adult). This suggests that changes in the regenerative capacity of frog nervous tissues during metamorphosis are due to changes other than ganglioside composition.