Review: Harmonised in vitro digestion and the Ussing chamber for investigating the effects of polyphenols on intestinal physiology in monogastrics and ruminants.

Because of the relevant effects of plant-derived polyphenols (PPs) on monogastrics and ruminants' nutrition, emissions and performance, an increasing number of in vivo and in vitro studies are being performed to better understand the mechanisms of action of polyphenols at both the ruminal and intestinal levels. The biological properties of these phenolic compounds strongly depend on their degradation, absorption and metabolism. The harmonised in vitro digestion method (INFOGEST) is one of the most reliable in vitro methods used to assess the bioaccessibility and or antioxidant activity of PP contained in different matrixes, as well as the interactions of PP and their degradation products with other feed ingredients. The effects of PP released from their matrix after in vitro digestion on different intestinal physiological parameters, such as epithelium integrity, can be further evaluated by the use of ex vivo models such as the Ussing chamber. This review aims to describe the combination of the INFOGEST method, coupled with the Ussing chamber as a valuable model for the digestion and subsequent effects and absorption of phenolic compounds in monogastrics and potentially in ruminants. The advances, challenges and limits of this approach are also discussed.

Effect of dietary L-carnitine supplementation to sows during gestation and/or lactation on sow productivity, muscle maturation and lifetime growth in progeny from large litters.

Genetic selection for increased sow prolificacy has resulted in decreased mean piglet birth-weight. This study aimed to investigate the effect of L-carnitine (CAR) supplementation to sows during gestation and/or lactation on sow productivity, semitendinosus muscle (STM) maturity, and lifetime growth in progeny. Sixty-four sows were randomly assigned to one of four dietary treatments at breeding until weaning; CONTROL (0mg CAR/d), GEST (125mg CAR/d during gestation), LACT (250mg CAR/d during lactation), and BOTH (125mg CAR/d during gestation & 250mg CAR/d during lactation). The total number of piglets born per litter was greater for sows supplemented with CAR during gestation (17.3 v 15.8 ± 0.52; P<0.05). Piglet birth-weight (total and live) was unaffected by sow treatment (P>0.05). Total myofibre number (P=0.08) and the expression level of selected myosin heavy chain genes in the STM (P<0.05) was greater in piglets of sows supplemented with CAR during gestation. Pigs from sows supplemented with CAR during gestation had lighter carcasses at slaughter than pigs from non-supplemented sows during gestation (83.8 v 86.7 ± 0.86kg; P<0.05). In conclusion, CAR supplementation during gestation increased litter size at birth without compromising piglet birth-weight. Results also showed that the STM of piglets born to sows supplemented with CAR during gestation was more developed at birth. However, carcass weight at slaughter was reduced in progeny of sows supplemented with CAR during gestation. The CAR supplementation strategy applied during gestation in this study could be utilized by commercial pork producers to increase sow litter size and improve offspring muscle development.

Pre-slaughter stress and horn status influence physiology and meat quality of young bulls.

The study used 81 young bulls, half of which were disbudded at 7 weeks of age. The effects of horn status during rearing and of acute physical and emotional stress just before slaughter on the physiological status at slaughter and subsequent meat quality were investigated. Bulls were reared in groups containing i) only bulls with horns, ii) only bulls without horns, or iii) mixed (half with, and half without horns). Bulls of each rearing condition were assigned to one of two slaughter conditions: with limited (LS) or with supplementary stress (SS). LS resulted in lower heart rates, stress hormone concentrations and carcass temperature, whereas SS resulted in faster post mortem pH decline and lower juiciness. Horned bulls from unmixed rearing groups had lower early pm temperature, shorter sarcomeres, and lower tenderness compared to disbudded bulls. Correlations and regression analysis revealed relationships between physiological indicators, mainly heart rate before slaughter, and meat quality, including water-holding capacity and indicators of proteolysis.

Influence of l-carnitine and l-arginine on protein synthesis and maturation of the semitendinosus muscle of lightweight piglets.

Lightweight (LW) piglets from large litters display impaired growth performance compared with heavier littermates. This study investigated the growth performance and muscle development of early-weaned LW piglets (birthweight <1.2 kg) from large litters (17.3 ± 3.0 total born per litter), fed ad libitum a milk replacer supplemented with either l-carnitine (CAR) or l-arginine (ARG) from day 7 to day 28 of age. In total, 36 female and entire male Swiss Large White piglets, weaned on day 7 of age, were artificially reared in pairs in rescue decks. They were allocated to one of three dietary treatments: unsupplemented control (CON), 0.48 g l-carnitine·piglet-1 ·day-1 (CAR) or 1.20 g l-arginine·kg body weight-1 ·day-1 (ARG). Milk replacer was prepared daily in a 1:4 powder-to-water ratio and fed ad libitum. Piglets were weighed at birth and on days 7, 14, 21 and 28. Feed intake was assessed daily. Piglets were euthanized on day 28. The entire semitendinosus muscle (STM) was collected, and organs were weighed. Subsequently, the STM was divided into the light (STMl ) and dark (STMd ) portion, and contractile and metabolic traits were analysed by ATP histochemistry, enzyme activities and gene expression. No differences in growth performance, organ and STM weight and on contractile traits were found between groups. A tendency (p < .10) for an elevated lipid oxidation enzyme activity in the STMl and STMd and greater (p < .05) phosphorylation of the mammalian target of rapamycin pathway in the STMl of CAR compared with CON piglets was found. Despite these metabolic responses, the lack of effect of CAR and ARG supplementation on growth performance suggests that providing the milk replacer ad libitum in combination with added CAR and ARG is insufficient for eliciting faster growth of LW piglets.

Milk replacers supplemented with either L-arginine or L-carnitine potentially improve muscle maturation of early reared low birth weight piglets from hyperprolific sows.

As a result of the selection for genotypes with greater sow prolificacy, litter size increased and, concomitantly, average litter birth weight and early postnatal survival rates of low birth weight (L-BtW) offspring decreased. This study compared the impact of l-carnitine (CAR) and l-arginine (ARG) supplemented with a milk replacer and fed to L-BtW piglets born from large litters from days 7 to 28 of age on growth performance, carcass composition, organ and Semitendinosus muscle (STM) development. A total of 30 female and castrated Swiss Large White piglets weaned at 7 days of age were assigned to three milk replacer diets containing either no supplement (CON), CAR (0.40 g/piglet per day) or ARG (1.08 g/kg BW per day). Piglets were kept in pairs in rescue decks (0.54 m2). They were weighed daily and daily allowance of both, feed and ARG, was adjusted accordingly. Thus, feed allowance depended on growth. Each day, the milk replacer was prepared with water (1:4). Feed (allowance: 60 g dry matter/kg BW per day) was offered daily in six equal rations. Feed intake and feed efficiency was assessed for the pairs and apparent total tract-energy and -protein digestibility was determined from days 21 to 28 of age. On day 28, piglets were euthanized, blood samples were collected and the whole STM and organs were weighed. In STM, the size and metabolic properties of myofibers were determined. No difference in growth performance was found between dietary treatments, but piglets from the CAR group tended (P<0.10) to grow faster during the 1st experimental week and consume more feed from days 14 to 21 as compared with piglets of the CON group. A setback in growth in the last week in the CAR group coincided with the lower (P<0.05) energy and protein digestibility. Dietary treatments had no effect on STM and organ weight and myofiber size. Compared with the other groups, there were trends (P<0.10) for blood serum urea and glucose level to be greater in CAR and for non-esterified fatty acid level to be greater in ARG piglets. The greater (P<0.05) ratio of lactate dehydrogenase to either citrate synthase or ß-hydroxyacyl-CoA dehydrogenase indicated that the relative importance of the glycolytic compared with the oxidative pathway was greater in STM of CAR and ARG compared with CON piglets. These results suggest that ARG and CAR supplements were beneficial for muscle maturation whereas findings on phenotypic traits were rather unsystematic.

Hydrolysable tannin-based diet rich in gallotannins has a minimal impact on pig performance but significantly reduces salivary and bulbourethral gland size.

Tannins have long been considered 'anti-nutritional' factors in monogastric nutrition, shown to reduce feed intake and palatability. However, recent studies revealed that compared with condensed tannins, hydrolysable tannins (HT) appear to have far less impact on growth performance, but may be inhibitory to the total activity of caecal bacteria. This in turn could reduce microbial synthesis of skatole and indole in the hindgut of entire male pigs (EM). Thus, the objective of this study was to determine the impact of a group of dietary HT on growth performance, carcass traits and boar taint compounds of group housed EM. For the study, 36 Swiss Large White boars were assigned within litter to three treatment groups. Boars were offered ad libitum one of three finisher diets supplemented with 0 (C), 15 (T15) or 30 g/kg (T30) of HT from day 105 to 165 of age. Growth performance, carcass characteristics, boar taint compounds in the adipose tissue and cytochrome P450 (CYP) isoenzymes CYP2E1, CYP1A2 and CYP2A19 gene expression in the liver was assessed. Compared with C, feed efficiency but not daily gain and daily feed intake was lower (P<0.05) in T15 and T30 boars. Except for the percentage carcass weight loss during cooling, which tended (P<0.10) to be greater in T30 than C and T15, carcass characteristics were not affected by the diets. In line with the numerically lower androstenone level, bulbourethral and salivary glands of T30 boars were lighter (P<0.05) than of T15 with intermediate values for C. Indole level was lower (P<0.05) in the adipose tissue of T30 than C pigs with intermediate levels in T15. Skatole levels tended (P<0.10) to be lower in T30 and C than T15 pigs. Hepatic gene expression of CYP isoenzymes did not differ between-treatment groups, but was negatively correlated (P<0.05) with androstenone (CYP2E1 and CYP1A2), skatole (CYP2E1, CYP2A) and indole (CYP2A) level. In line with the numerically highest androstenone and skatole concentrations, boar taint odour but not flavour was detected by the panellists in loins from T15 compared with loins from C and T30 boars. These results provide evidence that HT affected metabolism of indolic compounds and androstenone and that they affected the development of accessory sex glands. However, the effects were too small to be detected by sensory evaluation.

Effect of exchanging Onobrychis viciifolia and Lotus corniculatus for Medicago sativa on ruminal fermentation and nitrogen turnover in dairy cows.

The objective of the study was to determine the effect of feeding sainfoin (SF; Onobrychis viciifolia) and birdsfoot trefoil (BT; Lotus corniculatus), 2 temperate climate forage legumes that contain condensed tannins (CT), on ruminal fermentation and N turnover in dairy cows. Six ruminally cannulated multiparous dairy cows (milk yield=40kg/d; 36 d in milk) were used in a replicated 3×3 Latin square design. All animals were fed basal diets containing 20% pelleted SF (223g of CT/kg of dry matter), BT (30.3g of CT/kg of dry matter), or alfalfa (AL) and concentrate to meet their predicted nutrient requirements. Each experimental period consisted of a 21-d adaptation period in a tiestall, followed by a 7-d collection period in metabolic crates, where feces and urine were collected quantitatively. During the 7-d period, milk yield was recorded daily and milk samples were taken at each milking. Blood, ruminal fluid, and papillae were sampled on d 2 and 5. The relative abundance of selected bacterial strains in ruminal fluid and the gene expression of transporter genes in the papillae were determined with quantitative PCR. Total volatile fatty acids and the abundance of the cellulolytic bacteria Prevotella spp. and Ruminococcus flavefaciens decreased with SF compared with AL. The relative gene expression of the monocarboxylate transporter 1 was increased with BT compared with AL and SF. Total yields of milk, milk fat, and milk protein were similar among treatments. The proportion of 18:3n-3 in milk fat was greater and those of 22:5n-3 and 22:6n-3 were lower with SF than with BT. The contents of urea N in blood (2.71, 3.45, and 3.90mmol/L for SF, AL, and BT, respectively), milk (79.8, 100.1, and 110.9mg/kg for SF, AL, and BT, respectively), and urine were lower with SF than with AL and BT, and a trend toward a lower ruminal ammonia content occurred with SF compared with BT. Intake and excretion of N with milk were similar among treatments, but urine N was lower with SF than with AL. The N excretion to N intake relation showed a shift in a part of urine N (17.5, 20.8, and 19.5% for SF, AL, and BT, respectively) to fecal N (45.2, 41.3, and 38.5% for SF, AL, and BT respectively) with SF compared with AL and BT. In conclusion, SF and BT differed in their effects on fermentation and milk fatty acid profile and SF also showed potential to decrease metabolic and environmental loads. The main reason for the different efficiency was likely a higher CT content of SF compared with BT.

Urinary zearalenone measured with ELISA as a biomarker of zearalenone exposure in pigs.

The suitability to assess zearalenone (ZEA) exposure in pigs of a commercial ELISA kit for ZEA analysis in urine was tested. A daily dose of 0, 5, 10, 20 and 40 µg synthetic ZEA per kilogram BW was administered via the feed to four gilts per dose group, and after 3 and after 7 days of ZEA intake, urine samples were assayed with the ELISA which has a relative cross-reactivity of 42% with α-zearalenol. The concentration of urinary ZEA equivalents (ZEA plus 42% of α-zearalenol present) did not differ between day 4 and day 8 (P = 0.50) within each dose group. The urinary ZEA equivalent/creatinine ratio was tightly correlated with ZEA intake (r = 0.95). The urinary ZEA equivalent/creatinine values at 0 and 40 µg/kg BW were distinctly different from those of the intermediate dose levels, whereas there was some overlapping of the individual values at the dose levels 5, 10 and 20 µg/kg BW. The urinary ZEA equivalent/creatinine ratio can be used as a biomarker for ZEA exposure in pigs provided that urine samples of several animals receiving the same diet are assayed, either separately or after pooling.

Arterial biomechanics after destruction of cytoskeleton by Cytochalasin D.

Vascular smooth muscle is a major structural element of the arterial wall. We examined the effects of cytoskeleton destruction, after administration of Cytochalasin D, on the biomechanical properties of porcine common carotids. Compared to untreated, maximally dilated controls, Cytochalasin D-treated arteries have shown a marked increase in compliance in the elastin-dominated pressure range. After weakening the VSM stress-bearing cytoskeleton by Cytochalasin D the artery would expand, reaching a new equilibrium state. This study brings further evidence that VSM is under tension, even when it is under zero load and at maximal vasodilation. This residual tension was released upon partial destruction of the cytoskeleton with Cytochalasin D. From a biomechanical standpoint, this means that the zero stress states of the in-series and parallel elastic components are substantially different.

Oscillatory shear stress and reduced compliance impair vascular functions.

Shear stress has been shown to influence endothelial cell gene expression and morphology. In particular, low and bi-directional shear stress, mimicking conditions at plaque-prone areas, down-regulates the expression of several atheroprotective genes, and up-regulates that of other genes considered as pro-inflammatory. Another mechanical situation thought to have a negative influence on vascular functions is arterial stiffness. Loss of arterial compliance occurs during ageing, in diabetic as well as in hypertensive patients. In this work we investigated the effects of these two particular hemodynamic environments (bi-directional shear stress and reduced compliance), using a recently developed perfusion system allowing to expose native arteries in vitro to complex hemodynamic environments. We were able to show that both plaque-prone shear stress and reduced compliance trigger endothelial dysfunction, but via different mechanisms. Only reduced compliance affected vascular contractility, inducing a dedifferentiation of smooth muscle cells and a consequent loss of norepinephrine sensitivity.

Gelsolin superfamily proteins: key regulators of cellular functions.

Cytoskeletal rearrangement occurs in a variety of cellular processes and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins control actin organization by severing filaments, capping filament ends and nucleating actin assembly [1]. Gelsolin is the founding member of this family, which now contains at least another six members: villin, adseverin, capG, advillin, supervillin and flightless I. In addition to their respective role in actin filament remodeling, these proteins have some specific and apparently non-overlapping particular roles in several cellular processes, including cell motility, control of apoptosis and regulation of phagocytosis (summarized in table 1). Evidence suggests that proteins belonging to the gelsolin superfamily may be involved in other processes, including gene expression regulation. This review will focus on some of the known functions of the gelsolin superfamily proteins, thus providing a basis for reflection on other possible and as yet incompletely understood roles for these proteins.

Flow pulsatility is a critical determinant of oxidative stress in endothelial cells.

Atherosclerotic plaques are found in regions exposed to disturbed flow, suggesting the active participation of the hemodynamic environment in atherogenesis. Indeed, unidirectional and oscillatory flow patterns (ie, bidirectional) have been shown to induce contrasting effects on endothelial function. The purpose of the present study was to evaluate the effect of these 2 flow patterns characterizing plaque-free and plaque-prone regions, respectively, on the oxidative stress of endothelial cells. NADH-dependent oxidase activity was shown to be equally induced (2- to 3-fold) in endothelial cells exposed to pulsatile unidirectional or oscillatory flow patterns. Under these flow conditions, an increase in endothelial cell oxidative state compared with static cultures was observed. Pulsatility of flow, but not cyclic stretch, was a critical determinant of flow-induced superoxide anion production. P22phox mRNA level increased in cells exposed to both unidirectional and oscillatory shear stress, suggesting that p22phox gene expression upregulation contributes to flow-induced increase in superoxide anion production in endothelial cells. In conclusion, we demonstrate a flow-induced increase in oxidative stress in endothelial cells. This chronic increase is dependent on the pulsatile nature of flow and is mediated in part by upregulation of an NADH-dependent oxidase expression.

Unidirectional and oscillatory shear stress differentially modulate NOS III gene expression.

Atherosclerotic plaques preferentially develop in regions exposed to a low mean shear stress and cyclic reversal of flow direction (oscillatory flow). This contrasts with plaque-free zones where endothelial cells are exposed to unidirectional flow. Previous works from our laboratory using a unique experimental flow system demonstrated the existence of a differential regulation of endothelial nitric oxide synthase (NOS III) gene expression by unidirectional and oscillatory flow patterns. We further studied the possible mechanisms responsible for selective unresponsiveness of NOS III gene regulation to oscillatory flow. The results obtained demonstrate that (i) induction of the activity of the 1600-base-pair NOS III gene promoter by unidirectional and oscillatory shear stress is modulated by similar mechanisms that involve NF-kappaB activation, but do not involve Ras-dependent MAP kinase activation, and (ii) the lack of induction of NOS III gene regulation by oscillatory shear stress can be attributed to the activation of a yet unidentified negative cis-acting element present in the NOS III gene.

Nitric oxide synthase expression in endothelial cells exposed to mechanical forces.

Nitric oxide (NO) has been demonstrated to play a central role in vascular biology and pathobiology. The expression of endothelial NO synthase (eNOS) is regulated in part by blood flow-induced mechanical factors. The purpose of this study was to evaluate how the expression of eNOS mRNA correlates with the activation of its promoter in both arterial and venous endothelial cells (ECs) exposed to mechanical forces, ie, shear stress and cyclic circumferential stretch. Bovine aortic ECs (BAECs) and EA hy.926, a cell line derived from human umbilical vein ECs, were grown on the inside of elastic tubes and subjected to combinations of pressure, pulsatile shear stress, and cyclic circumferential stretch for 24 hours. Two patterns of shear stress were used: unidirectional (mean of 6, ranging from 3 to 9 dyne/cm2) and oscillatory (mean of 0.3, ranging from -3 to +3 dyne/cm2). The expression of eNOS mRNA was quantified by Northern blot analysis. Activation of the promoter was assessed by luciferase activity after the cells were transiently transfected before the flow experiments with a plasmid construct containing the fully functional eNOS promoter coupled to a luciferase reporter gene. Expression of eNOS mRNA was increased and promoter activity was enhanced by unidirectional shear stress compared with static control. Oscillatory shear slightly upregulated eNOS mRNA in BAECs, whereas it downregulated eNOS mRNA in EA hy.926. In both BAECs and EA hy.926, there was a good correlation between the increase in eNOS mRNA expression and promoter activation by unidirectional shear stress. In contrast, in both BAECs and EA hy.926 cells exposed to shear stress, cyclic stretch did not change eNOS mRNA expression, but the activation of eNOS promoter was significantly lower. Moreover, when ECs were exposed to oscillatory shear stress, there was a dramatic activation of the eNOS promoter. These results demonstrate that unidirectional shear stress increases eNOS mRNA expression via a transcriptional mechanism. However, oscillatory shear stress and cyclic stretch appear to control eNOS expression through posttranscriptional regulatory events.

Interleukin (IL)-6 and its soluble receptor induce TIMP-1 expression in synoviocytes and chondrocytes, and block IL-1-induced collagenolytic activity.

To define the potential role of interleukin-6 (IL-6) and its soluble receptor alpha in cartilage metabolism, we analyzed their effects on tissue inhibitor of metalloproteases (TIMP) synthesis by synoviocytes and chondrocytes. TIMP-1 production by isolated human articular synovial fibroblasts and chondrocytes, stimulated by IL-6 and/or its soluble receptor, was first assayed by specific enzyme-linked immunosorbent assay; the slight stimulatory effect of IL-6 on TIMP-1 production by both types of cells was markedly amplified by the addition of soluble receptor, the maximal secretion being observed only at 96 h. TIMP-1 mRNA expression, determined by ribonuclease protection assay, was induced by IL-6 together with its soluble receptor, but TIMP-2 and -3 mRNAs were not affected by these factors. A specific neutralizing antibody abolished the effects of the soluble receptor. Finally, supernatant from synoviocytes stimulated by IL-6 plus its soluble receptor blocked almost completely the collagenolytic activity of supernatant from IL-1-induced synoviocytes. These observations indicate that IL-6 and its soluble receptor have a protective role in the metabolism of cartilage. Given the high levels of soluble receptor in synovial fluid and the marked induction of IL-6 by IL-1 or TNF-alpha, it is likely that IL-6 and its soluble receptor are critical in controlling the catabolic effects of pro-inflammatory cytokines.

Endothelin-1 and endothelin-converting enzyme-1 gene regulation by shear stress and flow-induced pressure.

Hemodynamic forces have been shown to modulate the expression of endothelin (ET-1) and endothelin-converting enzyme (ECE-1) in endothelial cells. We have subjected E.A. hy 926 cells in culture to steady fluid shear stress with and without flow-induced pressure. The effect of combining these two mechanical forces on the expression of genes in the ET system was studied and the changes were compared to the mRNA levels in static culture. Analysis of total RNA by Northern blot analysis and RNAse protection showed that steady shear stress induced ET-1 gene expression three- to fourfold in this system. The same condition had little to no effect on altering expression of ECE-1 isoforms. A range of flow-induced pressure (80-160 mm Hg) was not able to further augment ET-1 or ECE-1 gene expression. Overall, with the mechanical environment studied, we have been able to detect a predominant contribution of shear stress to altering the ET-1 gene in our system. Furthermore, this induction was independent of an alteration of ECE-1 gene levels, suggesting that these two genes have a different pattern of regulation by the same stimuli in this cell type.

Concentrations and origins of soluble interleukin 6 receptor-alpha in serum and synovial fluid.

OBJECTIVE: To determine levels of soluble interleukin 6 receptor-alpha (sIL-6R alpha) in synovial fluid (SF) and serum from patients with different rheumatic diseases, and to analyze its cellular origin compared to IL-6. METHODS: IL-6 and sIL-6R alpha concentrations were measured in sera, SF, and culture supernatants of different cells types using specific sandwich ELISA. RESULTS: IL-6 levels were significantly higher (30 to 1000-fold) in SF than in sera, and higher in inflammatory arthropathies such as rheumatoid arthritis (RA), chondrocalcinosis, and gout than in osteoarthritis (OA). sIL-6R alpha levels in SF from patients with RA, gout, and chondrocalcinosis were also higher (24.7 +/- 7.5, 23.2 +/- 9.1, and 19.5 +/- 7.4 ng/ml, respectively) than in patients with OA (10.1 +/- 5 ng/ml), although the difference was distinctly smaller. In contrast, sIL-6R alpha concentrations did not differ significantly between the sera of healthy donors and patients. sIL-6R alpha levels were similar in SF and sera from inflammatory arthropathies, but lower in all osteoarthritic SF, compared to their corresponding serum. In contrast to IL-6, sIL-6R alpha was produced in high amounts by hepatocytes but not by structural cells of the joint (chondrocytes, synoviocytes, fibroblasts, and endothelial cells). Polymorphonuclear cells and mononuclear cells released intermediate levels. A significant correlation between sIL-6R alpha concentration and total number of leukocytes was observed in SF. CONCLUSION: Elevated levels of sIL-6R alpha were found in serum, likely to result from a marked release by hepatocytes in vitro. That levels are higher in inflammatory SF may be due in part to release by inflammatory cells in situ.

Tumor necrosis factor-alpha induction of major histocompatibility complex class II antigen expression is inhibited by interferon-gamma in a monocytic cell line.

Regulation of major histocompatibility complex (MHC) class II antigen expression by cytokines has been suggested to play a major role in the initiation and propagation of immune and autoimmune processes. The analysis of class II gene regulation benefits greatly from the existence of mutants with defects in regulatory factors. We report the establishment of a subclone of the human monocytic cell line U937, termed C119/9, with unusual cytokine regulation of MHC class II expression. In contrast to the parental U937 cell line, only tumor necrosis factor (TNF)-alpha, and not interferon (IFN)-gamma induces the expression of MHC class II antigens on C119/9 cells, and paradoxically, this induction was inhibited almost completely by IFN-gamma. The HLA-DR induction is controlled at the transcriptional level by the first 150 bp of the class II promoter which contains all the class II consensus elements. Both HLA-DR and -DQ mRNA are induced by TNF-alpha treatment, and both are diminished upon co-treatment with TNF-alpha and IFN-gamma. This antagonism between TNF-alpha and IFN-gamma seem to be restricted to MHC class II genes. This subline of U937 cells may be useful in further studies of MHC class II regulation.

Soluble interleukin-6 receptor strongly increases the production of acute-phase protein by hepatoma cells but exerts minimal changes on human primary hepatocytes.

Interleukin-6 (IL-6) interacts with a system of receptors, which include a 80-kDa IL-6-binding subunit (IL-6R) and a transducing element (gp130). The soluble form of IL-6R (sIL-6R) can bind its ligand and induce cellular responses by association with gp130, thus acting as an IL-6 agonist. We and others have previously shown that the responsiveness to IL-6 is different in hepatoma and human primary hepatocytes. We therefore compared the effects of sIL-6R on the two types of cells, and on the B9 hybridoma, another IL-6-sensitive cell line. Human primary hepatocytes, hepatoma cells PLC/PRF/5, and B9 cells were incubated with different concentrations of IL-6, sIL-6-R, or both. The hepatocyte culture supernatants were tested for their content of acute-phase proteins (APP). The proliferation of B9 cells was assessed by a colorimetric method. Results showed that sIL-6R alone markedly increased the production of APP by hepatoma cells in a dose-dependent manner, but affects only minimally primary hepatocytes and the proliferation of B9 cells. The combinations of IL-6R and its ligand enhanced the effects of Il-6 alone in both PLC/PRF/5 and B9 cells, but had no effect on primary hepatocytes. An immunohistochemical study indicated that the cell-surface expression of IL-6R was dramatically lower in hepatoma cells than in primary hepatocytes. In conclusion, our results show that the expression of IL-6R is low in the hepatoma cell PLC/PRF/5 when compared with primary hepatocytes and that this difference can, at least partly, explain their deficient responsiveness to IL-6. On the other hand, it appears that IL-6R expression by primary hepatocytes is sufficient and that circulating sIL-6R is unlikely to play a significant role in the modulation of IL6 effects.