Sentulic acid isolated from Sandoricum koetjape Merr attenuates lipopolysaccharide and interferon gamma co-stimulated nitric oxide production in murine macrophage RAW264 cells

A seco-triterpenoid, sentulic acid (SA) isolated from Sandoricum koetjape Merr attenuated nitric oxide (NO) production following co-stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in RAW264.7 macrophage cells. The mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), IFNγ, interleukin (IL)-6, and IL-12 in LPS/IFNγ co-stimulated RAW264.7 cells also decreased upon SA treatment. To determine the molecular mechanisms underlying the inhibitory effect of SA on LPS/IFNγ-induced NO production in RAW264.7 cells, we further analyzed Toll-like receptor (TLR) signaling by western blotting. The expression of TLR4 and IFN signaling molecules in cells treated with SA was significantly suppressed compared to that in cells not treated with SA. Additionally, SA inhibited the binding of LPS to the TLR4 receptor in RAW264.7 cells stimulated with Alexa Fluor 488-conjugated LPS. These results demonstrate that SA attenuates NO production after LPS/IFNγ co-stimulation in RAW264.7 cells by inhibiting the binding of LPS to TLR4. Our findings suggest that SA is beneficial for the treatment of inflammatory diseases.     

Potentiation of the T lymphocyte response to mitogens IV. Serum-free production and testing of macrophage soluble products.

Serum-free cultures of activated macrophages generate conditioned media containing both potentiating and inhibitory activities for the lectin-induced transformation of syngeneic thymocytes, lymph node cells, or spleen cells, also cultured in serum-free medium. Exhaustive dialysis of macrophage conditioned medium (MCM) eliminates its inhibitory activity. At mitogenic doses of lectin, the dialyzable material enhances the potentiating activity exerted by macro-molecular factors at low and optimal concentration of MCM. The inhibitory effect of intermediate concentrations of nondialyzed MCM on [³H]thymidine uptake can be reversed if the cells are washed and pulsed in fresh medium, and thus is artefactual. On the other hand, high doses have a real inhibitory effect on proliferative response of transformed lymphocytes. Rat MCM is not mitogenic for any of the target lymphocytes tested. Its effect is observed both in primary cultures of lymphocytes and secondary cultures of blast cells.     

Native IL-32 is released from intestinal epithelial cells via a non-classical secretory pathway as a membrane-associated protein

Although IL-32 has been shown to be induced under various pathological conditions, a detailed understanding of native IL-32 intracellular distribution and mechanism of release from cells has not been reported. We examined the expression of IL-32 in the intestinal epithelial cell line HT-29 following TNFα and IFNγ co-stimulation. The subcellular localization of induced IL-32 was associated with the membrane of lipid droplet-like structures and vacuolar structures that co-localized with markers of endosomes and lysosomes. Prolonged co-stimulation resulted in cell death and appearance of IL-32 in the culture medium. IL-32 released from co-stimulated HT-29 cells was found in a detergent-sensitive particulate fraction, and in a step density gradient the IL-32 particulate was buoyant, suggesting association with a membrane-bound vesicle. Upon Triton X-114 partitioning, most of the IL-32 partitioned to the detergent phase, suggesting hydrophobic characteristics. When IL-32-containing vesicles were subjected to protease K treatment, a protease resistant ～12kDa fragment was generated from ～24kDa IL-32. We propose that under these conditions, native IL-32 is released via a non-classical secretory route perhaps involving multi-vesicular bodies and exosomes. Demonstration of membrane association for both intracellular and released IL-32 suggests this unique cytokine may have a complex biosynthetic pathway and mechanism of action.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4⁺CD45RO⁺ T cells from peripheral blood, the percentage of ICOS⁺ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [³H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.     

Regulation of the formation of protease byBacillus megaterium

The formation of protease takes place in washed cells ofBacillus megaterium incubated in a nitrogen-free medium. The rate of enzyme synthesis is decreased much less than that of cell proteins as compared with growing cells. The synthesis of protease in a nitrogen-free medium requires the presence of glucose. The omission of glucose results in stopping of the enzyme formation and substantial decrease of the rate of protein synthesis. Protease is not synthesized when the washed cells are incubated in a phosphate, free medium. The incubation of the cells in a nitrogen-free medium results in a decrease of the concentration of amino acids in the pool. In a phosphate-free medium the content of free amino acids increases temporarily and decreases again later. When the culture grown in the medium containing threonine or threonine and isoleucine in addition to NH₄ ions is transferred into the medium without amino acids, no protease formation is found during derepression of enzymes synthesizing both amino acids. The cells grown in a medium containing casamino acids begin to form the enzyme after a short lag period when transferred into the medium containing NH₄ as a sole nitrogen source or into a nitrogen-free medium.     

Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning

We recorded neurons from the hippocampus of freely behaving rats during an auditory fear conditioning task. Rats received either paired or unpaired presentations of an auditory conditioned stimulus (CS) and an electric shock unconditioned stimulus (US). Hippocampal neurons (place and theta cells) acquired responses to the auditory CS in the paired but not in the unpaired group. After CS-US pairing, rhythmic firing of theta cells became synchronized to the onset of the CS. Conditioned responses of place cells were gated by their location-specific firing, so that after CS-US pairing, place cells responded to the CS only when the rat was within the cell's place field. These findings may help to elucidate how the hippocampus contributes to context-specific memory formation during associative learning.     

Testosterone and chemosensory detection in male Syrian hamster

Gonadal steroids stimulate both sexual motivation and performance. However, steroid facilitation of appetitive sexual behavior is poorly understood. The present study determined if castration impairs chemosensory detection in male hamsters. Chemosensory cues are the principal sensory modality to initiate mating in this species. We compared LiCl-induced conditioned taste avoidance to female hamster vaginal secretion (FHVS) in gonad-intact and castrated males. Following overnight water deprivation, males received FHVS for 15 min, followed by LiCl (2 ml of 0.15 M) or saline ip. The next day, fluid consumption in a two-bottle choice test was recorded for 5.5 h. Pairings were repeated 4×. Initially, discrimination of FHVS from estrous females (10 or 100 μg/ml) was compared with plain water. Subsequently, we determined if males could distinguish FHVS from Syrian vs. Djungarian females or from estrous vs. anestrous females. When 100 μg/ml FHVS was paired with saline, all gonad-intact and 86% of castrated males preferred FHVS over water. However, when 100 μg/ml FHVS was paired with LiCl, the preference was reversed: 12.5% of intact males and 25% of castrates preferred FHVS (P < 0.05 vs. saline pairing). When exposed to 10 μg/ml FHVS, neither gonad-intact nor castrated males expressed conditioned taste avoidance, suggesting that 10 μg/ml FHVS is below the threshold for detection. Comparing discrimination of FHVS from Syrian and Djungarian females, only castrated males developed a significant conditioned taste avoidance to Syrian FHVS paired with LiCl. While 71% of castrated males preferred Syrian FHVS after saline pairing, only 12.5% of castrates preferred Syrian FHVS after pairing with LiCl (P < 0.05). In gonad-intact males, 57% preferred Syrian FHVS after saline pairing, while 14% preferred Syrian FHVS following LiCl pairing (P > 0.05). Neither gonad-intact nor castrated males successfully discriminated between FHVS from estrous and anestrous females. These data demonstrate that castrated males perform as well as gonad-intact males in a test of LiCl-induced conditioned taste avoidance. Therefore, it is unlikely that steroids enhance detection of sexually relevant chemosensory cues.     

Neurite outgrowth induced by the substrate associated material from nonneuronal cells

Cultured embryonic heart cells release a powerful inducer of neurite outgrowth into the surrounding medium. The present report demonstrates that these cells also deposit material which induces neurite outgrowth directly onto their culture substratum. Thus, embryonic heart cells condition both the culture medium and the culture substratum with respect to neurite outgrowth. Conditioned substrata were prepared by incubating heart cell monolayers in EDTA until the cells released from the substratum and were discarded. When dissociated neurons from ciliary or sympathetic chain ganglia were plated in fresh medium onto a conditioned substratum, neurite outgrowth was initiated in 80–95% of the neurons within 60 min. The neurite-inducing activity is trypsin sensitive, but is not inactivated by antibodies to the cell attachment protein fibronectin, by the membrane-solubilizing detergent Triton X-100, or by the enzymes collagenase, RNase, or DNase. The factor in conditioned medium which also induces neurite outgrowth depends for its activity on attachment to an artificial polyornithine substratum, under which condition it appears to promote adhesion of neuronal filopodia to the substratum. Thus, neurite outgrowth in these two culture systems occurs only if the substratum is conditioned by the appropriate extracellular materials: conditioned either directly by the deposition of heart cell products or indirectly by the binding of a conditioned medium factor to the polyornithine substratum. These substratum-conditioning factors may be related to those components of the extracellular matrix which support neurite outgrowth in vivo.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-gamma and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4+CD45RO+ T cells from peripheral blood, the percentage of ICOS+ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [3H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-gamma, IL-4 and IL-10 in both SLE and normal T cells. IFN-gamma in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.     

Properties of olfactory conditioning in the housefly, Musca domestica

The housefly, Musca domestica, was conditioned to odours using the proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response and the properties of conditioning were investigated. Among trials including forward pairing of the conditioned stimulus (CS) with the unconditioned stimulus (US), backward pairing and isolated presentations of CS and US, only forward pairing is effective on the acquisition of conditioning. Backward pairing combined with forward pairing does not influence the effectiveness of the forward pairing. CS given overlapping with a US presentation permits only weak conditioning. The acquisition of conditioning decreases with increase of the CS-US interval. In the differential conditioning situation to two odours, discriminative responses are observed. In the flies conditioned with one antenna, the conditioned response is elicited not only by stimulation of the antenna used for conditioning but also by stimulation of the antenna not used for conditioning, although the response using the former is higher than with the latter. The ability to be conditioned is reduced immediately after fastening on a clay bed and increases with time. Ability can also be improved by transection of the ventral nerve cord.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium.The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.     

Mass selection of conditional mating mutants of Tetrahymena thermophila

The mating reaction in Tetrahymena thermophila includes a starvation period and two distinct cell interactions, co-stimulation and cell pairing, before the cells are cytoplasmically joined as conjugants. A selection procedure for harvesting mutants unable to mate at a restrictive temperature has been developed. A conjugant pair consisting of one cycloheximide-resistant cell and one wild-type cell (cycloheximide-sensitive) was itself sensitive to the drug. By adding cycloheximide and nutrient medium to a cross made at the restrictive and grow. Repetition of the selection procedure enriched for cells unable to conjugate at the restrictive temperature. The selected cells were able to grow at 38 degrees C and could conjugate at 28 degrees C. This procedure may be narrowed to select specifically for cell interaction mutants.     

The effects of N-methyl-N'-nitro-N-nitrosoguanidine on KB cells. II. Cell sensitivity to the lethal effect of the drug as a function of the cell age and of the nature of the culture medium

We have tested the sensitivity of KB cells to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), measured as cell loss within the 20-h period following a 1-h drug treatment, as a function of culture age and of the medium in which treated cells were incubated after elimination of MNNG. We showed that KB cell sensitivity to the lethal effect of the drug decreased with time after seeding when the treated cells were post-incubated in drug-free medium conditioned by untreated cells of the same age as treated ones but not when they were post-incubated in fresh drug-free medium. This difference was due in part to the fact that the conditioned medium had acquired with time a protective activity for treated cells and in part to an increased competence of aging cells to be protected by this medium. By post-incubating treated stationary cells sequentially in both media, we showed that a brief (15 min) post-incubation of the cells in fresh medium was sufficient to trigger cell death even if the cells were afterwards transferred to conditioned medium. In contrast, long post-incubation in fresh medium did not cause cell death if the cells were first post-incubated in conditioned medium for about 3 h. We conclude that: the medium acted on cell sensitivity to the lethal effect of MNNG through its growth regulatory ability; quiescent cells were less sensitive to the drug than growing cells; the sensitive phase of the cell was located before S; cell hypersensitivity might be due to deficient repair of cellular lesions rather than to increased lesion formation.     

Endogenous Encystment of Azotobacter vinelandii

When young cells of Azotobacter vinelandii are impinged on membrane filters, washed free of carbon substrate, and placed on a mineral salts basal medium, the culture will proceed to encyst although at a slower rate than if n-butanol were supplied as a substrate. The endogenous cysts are depleted in polyβ-hydroxybutyrate and have a narrower intine but show an increased resistance to desiccation and are susceptible to lysis by chelating agents. Membrane-supported cells reveal details of the encystment process such as the formation of a zone within the capsule prior to exine formation and the early deposition of exine structures.     

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. IV. The release of inhibitor from cultured cells.

Previous reports from our laboratory have shown that a culture medium, conditioned by the growth of isolated cells of Xenopus laevis blastulae, contains a low-molecular-weight substance which selectively inhibits 18 and 28S ribosomal RNA (rRNA) synthesis. Although the occurrence of an inhibitor in an acid-soluble fraction of blastulae has recently been demonstrated, our observation of an inhibitor in a conditioned medium has not been confirmed by other laboratories. To resolve this discrepancy, we have reexamined the effects of conditioned media and acid-soluble extracts on rRNA synthesis by neurula cells. (1) The inhibitory activity for rRNA synthesis can consistently be observed in blastula-conditioned media, provided some of the cells have been broken down during conditioning. If cell rupture is avoided, an inactive conditioned medium is obtained. (2) A homogenate of blastulae inhibits total RNA synthesis and shows no selective inhibition of rRNA synthesis. (3) Charcoal treatment of the conditioned medium and homogenate enhances their specificity for rRNA synthesis. It is then likely that cell breakdown may be involved in the release of the inhibitor into the medium and that some differences in the methods of preparation of conditioned medium may account for the above discrepancy.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Stimulation of plasmin activity in cultured human fibroblast cells by Porphyromonas endodontalis

• 1.1. Plasmin activity in the conditioned medium of Gin-1 cells, a human gingival fibroblast cell line, was stimulated by Porphyromonas endodontalis, a putative pathogen of oral submucous abscesses, in a time- and dose-dependent manner.
• 2.2. P. endodontalis stimulated the activity of plasminogen activator in both the conditioned medium and the cell lysate. The plasminogen activator in Gin-1 cells was approx. 50kDa by zymography.
• 3.3. The conditioned medium of Gin-1 cells exposed to P. endodontalis stimulated the conversion of human serum prekallikrein to kallikrein.
• 4.4. These results suggested that P. endodontalis stimulates the plasminogen activator-plasmin system in Gin-1 cells, and that activated plasmin plays a role in the progress of periodontal tissue inflammation.