Cat Heart Muscle in Vitro : VIII. Active transport of sodium in papillary muscles

The cells of cat right ventricular papillary muscles were depleted of K and caused to accumulate Na and water by preincubation at 2–3°C. The time courses of changes in cellular ion content and volume and of the resting membrane potential (V_m) were then followed after abrupt rewarming to 27–28°C. At physiological external K concentration ([K]_o = 5.32 mM) recovery of cellular ion and water contents was complete within 30 minutes, the maximal observable rates of K uptake and Na extrusion (Δmmol cell ion/(kg dry weight) (min.)) being 3.4 and 3.6, respectively. The recovery rate was markedly slowed at [K]_o = 1.0 mM. Rewarming caused V_m measured in cells at the muscle surface to recover within from <1 to 9 minutes, but only slight restoration of cellular ion contents (measured in whole muscles) had occurred after 10 minutes. Studies of recovery in NaCl-free sucrose Ringer''s solution made it possible to separate the ouabain-insensitive outward diffusion of Na as a salt from a simultaneous ouabain-sensitive Na extrusion which is associated with a net cellular K uptake. A hypothesis consistent with these observations is that rewarming may activate a ouabain-sensitive "electrogenic" mechanism, most probably the net active transport of Na out of the cell, from which net K uptake may then follow passively.     

Cat Heart Muscle in Vitro : VII. The temperature dependence of steady state K exchange in presence and absence of NaCl

In quiescent cat papillary muscles J _K, the rate of exchange of cellular K with K⁴² in the steady state, has been measured in the presence and absence of NaCl over a wide range of temperatures. J _K was found to be independent of the presence of external NaCl under the steady state conditions investigated. The Arrhenius plot for K exchange was linear over a range of temperatures from 2.5 to 37.5°C in the absence of NaCl, and from 17.5 to 37.5°C in the presence of NaCl. The corresponding apparent activation energies were, respectively, 10,200 and 8,800 calories/mole. J _K in the absence of NaCl was not affected by 10^-5 M ouabain. These results are consistent with a passive distribution for the K of heart muscle cells. The observations suggest that a carrier-mediated forced exchange of K for Na does not occur during the steady state in mammalian heart muscle.     

Cat Heart Muscle in Vitro : II. The steady state resting potential in quiescent papillary muscles

The steady state transmembrane resting potential difference (V_m) has been measured in quiescent papillary muscles. V_m was determined as a function of the external K concentration in Cl and SO₄ solutions and compared with the K equilibrium potential. Other measurements were made after replacement of external Na by choline, K by Rb and Cs, and Cl by SO₄, CH₃SO₄, and NO₃. Effects on V_m of albumin, temperature, and variation in internal K concentration are described.     

Cat Heart Muscle In Vitro : IV. Inhibition of transport in quiescent muscles

Cellular concentrations, [K]_i, [Na]_i, and [Cl]_i, and cell water contents were measured in vitro at 27°C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O₂, and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2–3°C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K]_i, was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K]_i remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O₂, or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.     

Cat Heart Muscle in Vitro : I. Cell volumes and intracellular concentrations in papillary muscle

Methods have been developed for the simultaneous determination of total water, inulin space, and K and Na content in muscles of 0.5 to 10 mg. wet weight. These methods have been used to define steady state conditions with respect to intracellular K concentration in papillary muscles from cat hearts perfused and contracting isometrically at 27–28°C. and at 37–38°C. Cell volumes and intracellular ionic concentrations have been followed as a function of the external K concentration and compared with values predicted on the basis of electroneutrality and osmotic equilibrium.     

Cat Heart Muscle in Vitro : III. The extracellular space

The "osmotic gradient" method, an intracellular microelectrode technique for determining whether an uncharged, water-soluble molecule enters cells or remains extracellular, is described. Using this method, a series of carbohydrates of graded molecular size were examined. In cat papillary muscles mannitol, molecular radius 4.0 Å, remained extracellular while arabinose, molecular radius 3.5 Å entered the cells. Measurement of the simultaneous uptake of H³-mannitol and C¹⁴-inulin showed that mannitol equilibrates with 40 per cent of total water in 1 hour, after which the mannitol space does not further increase. By contrast, inulin, molecular radius ~15 Å, equilibrates with 24 per cent of total water in 1 hour; thereafter the inulin space continues to increase very slowly. The intracellular K concentrations are significantly higher and the intracellular Na and Cl concentrations significantly lower when mannitol rather than inulin is used to measure the extracellular space. The intracellular Cl concentration determined with Cl³⁶ or Br⁸² is significantly higher than that calculated from the membrane potential assuming a passive Cl distribution. In addition, it is shown that choline enters and is probably metabolized by the cells of papillary muscle.     

Engineered Heart Tissue: A Novel Tool to Study the Ischemic Changes of the Heart In Vitro

Background

Understanding the basic mechanisms and prevention of any disease pattern lies mainly on development of a successful experimental model. Recently, engineered heart tissue (EHT) has been demonstrated to be a useful tool in experimental transplantation. Here, we demonstrate a novel function for the spontaneously contracting EHT as an experimental model in studying the acute ischemia-induced changes in vitro.

Methodology/Principal Findings

EHT was constructed by mixing cardiomyocytes isolated from the neonatal rats and cultured in a ring-shaped scaffold for five days. This was followed by mechanical stretching of the EHT for another one week under incubation. Fully developed EHT was subjected to hypoxia with 1% O₂ for 6 hours after treating them with cell protective agents such as cyclosporine A (CsA) and acetylcholine (ACh). During culture, EHT started to show spontaneous contractions that became more synchronous following mechanical stretching. This was confirmed by the increased expression of gap junctional protein connexin 43 and improved action potential recordings using an optical mapping system after mechanical stretching. When subjected to hypoxia, EHT demonstrated conduction defects, dephosphorylation of connexin-43, and down-regulation of cell survival proteins identical to the adult heart. These effects were inhibited by treating the EHT with cell protective agents.

Conclusions/Significance

Under hypoxic conditions, the EHT responds similarly to the adult myocardium, thus making EHT a promising material for the study of cardiac functions in vitro.     

Cat Heart Muscle in Vitro : IX. Cell ion and water contents in anisosmolal solutions

Cell contents of water, K, Na, and Cl have been determined in cat right ventricular papillary muscles immersed in solutions with and without NaCl when the external osmolality was varied with sucrose. The plot of cell water/kilogram dry weight (corrected for sucrose content) vs. (external osmolality)^-1 suggests that not less than 82% of water present in cells at physiological external osmolality is free to move across the cell membrane in response to an imposed osmotic gradient. Cells fail to increase their water content in very hypotonic solutions. For osmolalities greater than 5 times isosmolal, at which the mannitol space and the Cl³⁶ space are both equal to 100% of muscle water, rather large amounts of univalent cation appear to remain "bound" to the tissue.     

In Vivo Studies on Fast and Slow Muscle Fibers in Cat Extraocular Muscles

In anesthetized in vivo preparations, responses of two types of extraocular muscle fibers have been studied. The small, multiply innervated slow fibers have been shown to be capable of producing propagated impulses, and thus have been labeled slow multi-innervated twitch fibers. Fast and slow multi-innervated twitch fibers are distinguished by impulse conduction velocities, by ranges of membrane potentials, by amplitudes and frequencies of the miniature end plate potentials, by responses to the intravenous administration of succinylcholine, by the frequency of stimulation required for fused tetanus, and by the velocities of conduction of the nerve fibers innervating each of the muscle fiber types.     

Effects of Prestretch on Neonatal Peripheral Nerve: An In Vitro Study

Background  Characterizing the biomechanical failure responses of neonatal peripheral nerves is critical in understanding stretch-related peripheral nerve injury mechanisms in neonates. Objective  This in vitro study investigated the effects of prestretch magnitude and duration on the biomechanical failure behavior of neonatal piglet brachial plexus (BP) and tibial nerves. Methods  BP and tibial nerves from 32 neonatal piglets were harvested and prestretched to 0, 10, or 20% strain for 90 or 300 seconds. These prestretched samples were then subjected to tensile loading until failure. Failure stress and strain were calculated from the obtained load-displacement data. Results  Prestretch magnitude significantly affected failure stress but not the failure strain. BP nerves prestretched to 10 or 20% strain, exhibiting significantly lower failure stress than those prestretched to 0% strain for both prestretch durations (90 and 300 seconds). Likewise, tibial nerves prestretched to 10 or 20% strain for 300 seconds, exhibiting significantly lower failure stress than the 0% prestretch group. An effect of prestretch duration on failure stress was also observed in the BP nerves when subjected to 20% prestretch strain such that the failure stress was significantly lower for 300 seconds group than 90 seconds group. No significant differences in the failure strains were observed. When comparing BP and tibial nerve failure responses, significantly higher failure stress was reported in tibial nerve prestretched to 20% strain for 300 seconds than BP nerve. Conclusion  These data suggest that neonatal peripheral nerves exhibit lower injury thresholds with increasing prestretch magnitude and duration while exhibiting regional differences.     

Cation Transport in Escherichia coli : VI. K exchange

K influx and net K flux have been measured in suspensions of chloramphenicol-arrested Escherichia coli. The rate of K exchange in the steady state was independent of the K concentration of the medium over a 200-fold range. Under a number of experimental conditions the rate of exchange may be considerably increased or decreased without changing the cellular K content. These results show that under these conditions changes in K influx are associated with equal changes in K efflux, and suggest that the latter process is, at least in part, both carrier-mediated and tightly coupled to the influx process.     

Subunit and Catalytic Component Stoichiometries of an in Vitro Reconstituted Human Pyruvate Dehydrogenase Complex

The human pyruvate dehydrogenase complex (PDC) is a 9.5-megadalton catalytic machine that employs three catalytic components, i.e. pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), to carry out the oxidative decarboxylation of pyruvate. The human PDC is organized around a 60-meric dodecahedral core comprising the C-terminal domains of E2p and a noncatalytic component, E3-binding protein (E3BP), which specifically tethers E3 dimers to the PDC. A central issue concerning the PDC structure is the subunit stoichiometry of the E2p/E3BP core; recent studies have suggested that the core is composed of 48 copies of E2p and 12 copies of E3BP. Here, using an in vitro reconstituted PDC, we provide densitometry, isothermal titration calorimetry, and analytical ultracentrifugation evidence that there are 40 copies of E2p and 20 copies of E3BP in the E2p/E3BP core. Reconstitution with saturating concentrations of E1p and E3 demonstrated 40 copies of E1p heterotetramers and 20 copies of E3 dimers associated with the E2p/E3BP core. To corroborate the 40/20 model of this core, the stoichiometries of E3 and E1p binding to their respective binding domains were reexamined. In these binding studies, the stoichiometries were found to be 1:1, supporting the 40/20 model of the core. The overall maximal stoichiometry of this in vitro assembled PDC for E2p:E3BP:E1p:E3 is 40:20:40:20. These findings contrast a previous report that implicated that two E3-binding domains of E3BP bind simultaneously to a single E3 dimer (Smolle, M., Prior, A. E., Brown, A. E., Cooper, A., Byron, O., and Lindsay, J. G. (2006) J. Biol. Chem. 281, 19772–19780).The human pyruvate dehydrogenase complex (PDC)³ resides in mitochondria and catalyzes the oxidative decarboxylation of pyruvate to yield acetyl-CoA and reducing equivalents (NADH), serving as a link between glycolysis and the Krebs cycle (1–3). The PDC is a large (∼9.5 MDa) catalytic machine comprising multiple protein components. The three catalytic components are pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3), with E3 being a common component between different α-keto acid dehydrogenase complexes. The two regulatory enzymes in the PDC are the isoforms of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.The PDC is organized around a structural core, which includes the C-terminal domains of E2p and a noncatalytic component that specifically binds E3, i.e. the E3-binding protein (E3BP). To this E2p/E3BP core, multiple copies of the other PDC components are tethered through noncovalent interactions. Each E2p subunit contains two consecutive N-terminal lipoic acid-bearing domains (LBDs), termed L1 and L2, followed by the E1p-binding domain (E1pBD) and the C-terminal inner-core/catalytic domain, with these independent domains connected by unstructured linkers. Similarly, each E3BP subunit consists of a single N-terminal LBD (referred to as L3), the E3-binding domain (E3BD), and the noncatalytic inner core domain. Together, the inner core domains of E2p and E3BP assemble to form the dodecahedral 60-meric E2p/E3BP core. The role of the E1pBD and E3BD domains is to tether E1p and E3, respectively, to the periphery of the E2p/E3BP core. It is presumed that the LBDs (L1, L2, and L3) shuttle between the active sites of the three catalytic components of the PDC during the oxidative decarboxylation cycle (4). The eukaryotic PDC is unique among α-keto acid dehydrogenase complexes in its requirement for E3BP; prokaryotic PDCs employ the single subunit-binding domain to secure either E1p or E3 to the complex (5).Using a “divide-and-conquer” approach, a wealth of structural information on the PDC has been accumulated recently. High-resolution crystal structures are available for the human E1p (6–8) and E3 components (9). A model for the human E2p has been constructed based on an 8.8-Å electron density map available from cryo-electron microscopy (10). Additionally, solution and crystal structures of the L1 and L2 domains of E2p have been determined (11–13), and the high-resolution crystal structures of the E3BD (14, 15), pyruvate dehydrogenase kinase isoforms 1–4 (12, 16–18), and pyruvate dehydrogenase phosphatase isoform 1 (19) are known. Therefore, atomic models are available for almost all components and domains of the mammalian PDC.With the successes of the above structural approach, attention has turned to the overall structure of the PDC. There are two outstanding questions as follows. What are the subunit and overall catalytic component stoichiometries? What are the positions and orientations of the components in this large catalytic machine? Yu et al. (10) recently determined the cryo-EM structure of a PDC core comprising only human E2p subunits. Like yeast E2p, human E2p adopts a dodecahedral structure composed of 60 E2p proteins; each face of the dodecahedron has a large gap. Although this structure is highly informative, the composition of this core deviates substantially from that of the native PDC, because no E3BP subunits are present in the core structure. Based on the similar structure of the dodecahedral yeast PDC, a hypothesis was formed that, in human PDC, 12 copies of E3BP bind in the 12 gaps, which is termed the “60/12” model (20). Biophysical studies on complexes of E2p and E3BP later negated the 60/12 model; Hiromasa et al. (21) therefore posited an alternative, the “48/12” model, in which the dodecahedral core includes 48 E2p subunits and 12 E3BP proteins. A further source of conjecture is how many E1p and E3 components bind to the periphery of the PDC. If one binding domain binds to one peripheral catalytic component, a maximally occupied 60/12 PDC would harbor 60 E1p heterotetramers and 12 E3 dimers (or 48 E1ps and 12 E3s in the 48/12 model). The notion of such 1:1 binding is supported by the preponderance of available biophysical evidence. Specifically, two crystal structures, site-directed mutagenesis, and calorimetric measurements describe a 1:1 interaction between E3BD and E3 (14, 15). Also, although no structures are available for the human E1p-E1pBD complex, a crystal structure of the homologs of these proteins from Bacillus stearothermophilus also demonstrates a 1:1 interaction between the E1pBD of E2p and the E1p heterotetramer (22). In addition, ITC experiments performed on the bacterial E1p and the cognate subunit-binding domain indicate a 1:1 association (23). At variance with the above observations, a different subunit stoichiometry has been proposed by Smolle et al. (24, 25). Their evidence suggests that two binding domains bind for every peripheral component; such an arrangement potentially yields a PDC with half as many peripheral components bound.This study was undertaken to ascertain the subunit and component stoichiometries of the human PDC, particularly with regard to interactions between the E3BD and the E3 dimer. We show that quantification of bands on an SDS-polyacrylamide gel of a PDC reconstituted at saturating E1p and E3 concentrations supports neither the 60/12 nor the 48/12 model. Instead, a “40/20” model is proposed, and subsequent ITC and analytical ultracentrifugation (AUC) data corroborate this new model. In addition, results from electrophoretic mobility shift assays, ITC, and AUC presented here uniformly show a 1:1 interaction between E3BD and the E3 dimer as well as between E1pBD and the E1p heterotetramer. The implications of this 1:1 binding stoichiometry for the macromolecular assembly of the PDC are discussed.     

Amyloid Precursor Protein Mediated Changes in Intestinal Epithelial Phenotype In Vitro

Background

Although APP and its proteolytic metabolites have been well examined in the central nervous system, there remains limited information of their functions outside of the brain. For example, amyloid precursor protein (APP) and amyloid beta (Aβ) immunoreactivity have both been demonstrated in intestinal epithelial cells. Based upon the critical role of these cells in absorption and secretion, we sought to determine whether APP or its metabolite amyloid β (Aβ), had a definable function in these cells.

Methodology/Principal Findings

The human colonic epithelial cell line, Caco-2 cells, were cultured to examine APP expression and Aβ secretion, uptake, and stimulation. Similar to human colonic epithelium stains, Caco-2 cells expressed APP. They also secreted Aβ 1-40 and Aβ 1-42, with LPS stimulating higher concentrations of Aβ 1-40 secretion. The cells also responded to Aβ 1-40 stimulation by increasing IL-6 cytokine secretion and decreasing cholesterol uptake. Conversely, stimulation with a sAPP-derived peptide increased cholesterol uptake. APP was associated with CD36 but not FATP4 in co-IP pull down experiments from the Caco-2 cells. Moreover, stimulation of APP with an agonist antibody acutely decreased CD36-mediated cholesterol uptake.

Conclusions/Significance

APP exists as part of a multi-protein complex with CD36 in human colonic epithelial cells where its proteolytic fragments have complex, reciprocal roles in regulating cholesterol uptake. A biologically active peptide fragment from the N-terminal derived, sAPP, potentiated cholesterol uptake while the β secretase generated product, Aβ1-40, attenuated it. These data suggest that APP is important in regulating intestinal cholesterol uptake in a fashion dependent upon specific proteolytic pathways. Moreover, this biology may be applicable to cells beyond the gastrointestinal tract.     

Bacterial Cellulose-Binding Domain Modulates in Vitro Elongation of Different Plant Cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 μg mL⁻¹ CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 μg mL⁻¹, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.     

Alteration of Antioxidant Enzymes and Associated Genes Induced by Grape Seed Extracts in the Primary Muscle Cells of Goats In Vitro

This study was conducted to investigate how the activity and expression of certain paramount antioxidant enzymes respond to grape seed extract (GSE) addition in primary muscle cells of goats. Gluteal primary muscle cells (PMCs) isolated from a 3-week old goat were cultivated as an unstressed cell model, or they were exposed to 100 µM H₂O₂ to establish a H₂O₂-stimulated cell model. The activities of catalase (CAT), superoxide dismutases (SOD) and glutathione peroxidases (GPx) in combination with other relevant antioxidant indexes [i.e., reduced glutathione (GSH) and total antioxidant capacity (TAOC)] in response to GSE addition were tested in the unstressed and H₂O₂-stimulated cell models, and the relative mRNA levels of the CAT, GuZu-SOD, and GPx-1 genes were measured by qPCR. In unstressed PMCs, GSE addition at the dose of 10 µg/ml strikingly attenuated the expression levels of CAT and CuZn-SOD as well as the corresponding enzyme activities. By contrast, in cells pretreated with 100 µM H₂O₂, the expression and activity levels of these two antioxidant enzymes were enhanced by GSE addition at 10 µg/ml. GSE addition promoted GPx activity in both unstressed and stressed PMCs, while the expression of the GPx 1 gene displayed partial divergence with GPx activity, which was mitigated by GSE addition at 10 µg/ml in unstressed PMCs. GSH remained comparatively stable except for GSE addition to H₂O₂-stimulated PMCs at 60 µg/ml, in which a dramatic depletion of GSH occurred. Moreover, GSE addition enhanced TAOC in unstressed (but not H₂O₂-stimulated) PMCs. GSE addition exerted a bidirectional modulating effect on the mRNA levels and activities of CAT and SOD in unstressed and stressed PMCs at a moderate dose, and it only exhibited a unidirectional effect on the promotion of GPx activity, reflecting its potential to improve antioxidant protection in ruminants.     

Triclabendazole Sulfoxide Causes Stage-Dependent Embryolethality in Zebrafish and Mouse In Vitro

Background

Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic diseases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on embryonic development have been scarcely studied, and it has not been assigned a pregnancy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during gestation is needed.

Methodology

The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabendazole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfoxide were included as positive controls.

Principal Findings

Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and triclabendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h of exposure in rodent embryos and zebrafish (lowest observed adverse effect concentrations = 10 μM).

Conclusions/Significance

In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg), the main compound found in plasma is triclabendazole sulfoxide (maximum concentration 38.6 μM), while triclabendazole concentrations are approximately 30 times lower (1.16 μM). From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the organogenesis period, but its first metabolite triclabendazole sulfoxide has a high embryotoxic capacity in vitro during the preimplantation stage.     

The Induction of Lipocalin-2 Protein Expression in Vivo and in Vitro

Lipocalin-2 (LCN2) is secreted from adipocytes, and its expression is up-regulated in obese and diabetic mice and humans. LCN2 expression and secretion have been shown to be induced by two proinflammatory cytokines, IFNγ and TNFα, in cultured murine and human adipocytes. In these studies, we demonstrated that IFNγ and TNFα induced LCN2 expression and secretion in vivo. Although we observed a strong induction of LCN2 expression and secretion from white adipose tissue (WAT) depots, the induction of LCN2 varied among different insulin-sensitive tissues. Knockdown experiments also demonstrated that STAT1 is required for IFNγ-induced lipocalin-2 expression in murine adipocytes. Similarly, knockdown of p65 in adipocytes demonstrated the necessity of the NF-κB signaling pathway for TNFα-mediated effects on LCN2. Activation of ERKs by IFNγ and TNFα also affected STAT1 and NF-κB signaling through modulation of serine phosphorylation. ERK activation-induced serine phosphorylation of both STAT1 and p65 mediated the additive effects of IFNγ and TNFα on LCN2 expression. Our results suggest that these same mechanisms occur in humans as we observed STAT1 and NF-κB binding to the human LCN2 promoter in chromatin immunoprecipitation assays performed in human fat cells. These studies substantially increase our knowledge regarding the requirements and mechanisms used by proinflammatory cytokines to induce LCN2 expression.     

Lactobacilli Regulate Staphylococcus aureus 161:2-Induced Pro-Inflammatory T-Cell Responses In Vitro

There seems to be a correlation between early gut microbiota composition and postnatal immune development. Alteration in the microbial composition early in life has been associated with immune mediated diseases, such as autoimmunity and allergy. We have previously observed associations between the presence of lactobacilli and Staphylococcus (S.) aureus in the early-life gut microbiota, cytokine responses and allergy development in children. Consistent with the objective to understand how bacteria modulate the cytokine response of intestinal epithelial cell (IEC) lines and immune cells, we exposed IEC lines (HT29, SW480) to UV-killed bacteria and/or culture supernatants (-sn) from seven Lactobacillus strains and three S. aureus strains, while peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated by bacteria-sn or with bacteria conditioned IEC-sn. Although the overall IEC response to bacterial exposure was characterized by limited sets of cytokine and chemokine production, S. aureus 161:2-sn induced an inflammatory response in the IEC, characterized by CXCL1/GROα and CXCL8/IL-8 production, partly in a MyD88-dependent manner. UV-killed bacteria did not induce a response in the IEC line, and a combination of both UV-killed bacteria and the bacteria-sn had no additive effect to that of the supernatant alone. In PBMC, most of the Lactobacillus-sn and S. aureus-sn strains were able to induce a wide array of cytokines, but only S. aureus-sn induced the T-cell associated cytokines IL-2, IL-17 and IFN-γ, independently of IEC-produced factors, and induced up regulation of CTLA-4 expression and IL-10 production by T-regulatory cells. Notably, S. aureus-sn-induced T-cell production of IFN- γ and IL-17 was down regulated by the simultaneous presence of any of the different Lactobacillus strains, while the IEC CXCL8/IL-8 response was unaltered. Thus these studies present a possible role for lactobacilli in induction of immune cell regulation, although the mechanisms need to be further elucidated.