Function and molecular modeling of the interaction between human interleukin 6 and its HNK-1 oligosaccharide ligands.

Interleukin 6 (IL-6) is endowed with a lectin activity for oligosaccharide ligands possessing the HNK-1 epitope (3-sulfated glucuronic acid) found on some mammalian glycoprotein N-glycans (Cebo, C., Dambrouck, T., Maes, E., Laden, C., Strecker, G., Michalski, J. C., and Zanetta, J. P. (2001) J. Biol. Chem. 276, 5685-5691). Using high affinity oligosaccharide ligands, it is demonstrated that this lectin activity is responsible for the early dephosphorylation of tyrosine residues found on specific proteins induced by interleukin 6 in human resting lymphocytes. The gp130 glycoprotein, the signal-transducing molecule of the IL-6 pathway, is itself a molecule possessing the HNK-1 epitope. This indicates that IL-6 is a bi-functional molecule able to extracellularly associate its alpha-receptor with the gp130 surface complex. Computational modeling indicates that the lower energy conformers of the high affinity ligands of IL-6 have a common structure. Docking experiments of these conformers suggest that the carbohydrate recognition domain of IL-6 is localized in the domain previously identified as site 3 of IL-6 (Somers, W., Stahl, M., and Seehra, J. S. (1997) EMBO J. 16, 989-997), already known to be involved in interactions with gp130.     

Pullulan-cyclodextrin microspheres. A chromatographic approach for the evaluation of the drug-cyclodextrin interactions and the determination of the drug release profiles

Pullulan microspheres containing cyclodextrin (CyD) were obtained by chemical crosslinking with epichlorohydrin of an alkaline solution of pullulan (Pul) and alpha-, beta- or gamma-CyD. The amount of alpha-, beta- and gamma-CyD in microspheres was 120, 156, and 138 micromol/g, respectively, as determined from the percentage of iodine incorporated in the hydrophobic cavity of CyD's. Microspheres were packed in a glass column and the liquid chromatographic behaviour by isocratic elution of different drugs or typical organic compounds (TOC), taken as model drugs, was investigated. The increase of the retention volume (V(R)) of each compound, depending on the interaction(s) between CyD's cavity and the considered molecule, is characterized by a broadening of the peaks. The interaction coefficient K, corresponding to the ratio between the V(R) value of each tested molecule on Pul-alpha-, Pul-beta- and Pul-gamma-CyD active stationary phase and the V(R) value of benzoic acid on St/maltodextrin neutral stationary phase, was determined. According to K values, the accurate prediction can be done on the potential drugs to be conditioned in suitable CyD cavity. Values of K allow to anticipate the release profiles of drugs considered.     

Effects of fast neutrons on chromatin: dependence on chromatin structure

The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin - proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)--heterochromatin (compact) ratio.     

Lymphoma immunophenotyping: "borderline" lymphomas

Immunophenotyping of B-cell lymphoproliferative disorders is indispensable, especially in disorders with CD19(+) CD5(+) B lymphocytes, where we have to make the distinction between low grade neoplasia, such as chronic lymphocytic leukemia with CD23(+) malignant lymphocytes, and aggressive neoplasia such as mantle cell lymphoma with CD23(-) malignant lymphocytes. We found some cases of CD19(+) CD5(+) lymphoproliferative disorders that do not meet all criteria for diagnosis of chronic lymphocytic leukemia or mantle cell lymphoma. For instance, we found cases with a low or no expression of CD23, asociated with absence of expression of FMC7 and surface immunoglobulins. These cases could be classified as "borderline" CD19(+) CD5(+) B cell lymphoproliferative disorders, with an intermediate neoplasic grade.     

The neuropeptide vasoactive intestinal peptide generates tolerogenic dendritic cells

Tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction/activation of regulatory T cells (Treg). Endogenous factors contribute to the functional development of tolerogenic DCs. In this report, we present evidence that two known immunosuppressive neuropeptides, the vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), contribute to the development of bone marrow-derived tolerogenic DCs in vitro and in vivo. The VIP/PACAP-generated DCs are CD11c(low)CD45RB(high), do not up-regulate CD80, CD86, and CD40 following LPS stimulation, and secrete high amounts of IL-10. The induction of tolerogenic DCs is mediated through the VPAC1 receptor and protein kinase A, and correlates with the inhibition of IkappaB phosphorylation and of NF-kappaBp65 nuclear translocation. The VIP/PACAP-generated DCs induce functional Treg in vitro and in vivo. The VIP/DC-induced Treg resemble the previously described Tr1 in terms of phenotype and cytokine profile, suppress primarily Th1 responses including delayed-type hypersensitivity, and transfer suppression to naive hosts. The effect of VIP/PACAP on the DC-Treg axis represents an additional mechanism for their general anti-inflammatory role, particularly in anatomical sites which exhibit immune deviation or privilege.     

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE-Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2'-7' dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE-Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. A three times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro-diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.     

An information-theoretic approach to curiosity-driven reinforcement learning

We provide a fresh look at the problem of exploration in reinforcement learning, drawing on ideas from information theory. First, we show that Boltzmann-style exploration, one of the main exploration methods used in reinforcement learning, is optimal from an information-theoretic point of view, in that it optimally trades expected return for the coding cost of the policy. Second, we address the problem of curiosity-driven learning. We propose that, in addition to maximizing the expected return, a learner should choose a policy that also maximizes the learner's predictive power. This makes the world both interesting and exploitable. Optimal policies then have the form of Boltzmann-style exploration with a bonus, containing a novel exploration-exploitation trade-off which emerges naturally from the proposed optimization principle. Importantly, this exploration-exploitation trade-off persists in the optimal deterministic policy, i.e., when there is no exploration due to randomness. As a result, exploration is understood as an emerging behavior that optimizes information gain, rather than being modeled as pure randomization of action choices.     

In vitro differentiation of dendritic cells in the presence of prostaglandin E2 alters the IL-12/IL-23 balance and promotes differentiation of Th17 cells

PGE2, an endogenous lipid mediator released in inflammatory conditions, affects both dendritic cell (DC) differentiation and maturation. Whereas the effect of PGE2 on fully differentiated DC was studied extensively, little is known about its effects on DC differentiation. In this study, we show that bone marrow-derived DC generated in the presence of PGE2 (DCp) acquire a proinflammatory profile; produce higher levels of proinflammatory cytokines/chemokines; express higher levels of MHC class II, costimulatory molecules, and TLRs; and exhibit increased activation of the NF-kappaB-signaling pathway. In addition, DCp exhibit a different IL-12/IL-23 profile than DC generated in the absence of PGE2. The low IL-12 and high IL-23 production in LPS-stimulated DCp is associated with the down-regulation of p35 and the up-regulation of p19 expression, respectively. In agreement with the DCp proinflammatory phenotype and especially with the altered IL-12/IL-23 balance which strongly favors IL-23, DCp also affect T cell differentiation. In contrast to DC which favor Th1 differentiation, DCp promote Th17 and inhibit Th1/Th2 differentiation, in vitro and in vivo. Previous in vivo studies indicated that PGE2 had a proinflammatory effect, especially in models of autoimmune diseases. Our results suggest that the proinflammatory effects of PGE2 could be mediated, at least partially, through effects on differentiating DC and subsequent alterations in CD4+ T cell differentiation, resulting in the preferential development of pathogenic autoimmune Th17 cells.     

Binding is not enough: flexibility is needed for photocrosslinking of Lck kinase by benzophenone photoligands

Benzophenone photophores are employed widely for photoaffinity-labeling studies. Photolabeling with benzophenone, however, is hardly a routine experiment. Even when a photoprobe binds to its target, photocrosslinking does not necessarily occur. This is because photolabeling by benzophenone is affected by many factors other than target-binding, such as conformational flexibility of photoligand. Despite the widespread recognition of such complications, there has been no systematic study to assess the relative importance of individual factors that can affect photolabeling efficiency. In order to gain an insight into this problem, we conducted a structure-activity relationship (SAR) study of benzophenone photoligands for Lck kinase, in which photoligands with varying target-binding affinity and conformational flexibility were compared. The study found that binding-affinity, as indicated by kinase inhibitory potency, did not correlate with photolabeling efficiency. Instead, conformational flexibility was found to be the determining factor for efficient photolabeling by our photoligands. Implication of the current findings, in particular, with regard to selection and optimization of benzophenone photoligands, is discussed.