Morphine attenuates the expression of sensitization to ethanol, but opioid antagonists do not

Behavioral sensitization to ethanol is characterized by an increased locomotor activity after repeated exposure. A great variability exists among species and strains in the development of sensitization. There is a growing amount of evidence to indicate that the opioid system is involved in alcoholism; it is possible, therefore, that this system also modulates the sensitization to ethanol. In this study we evaluated the role of the opioid system in determining the variability of the sensitized response to ethanol. Mice received repeated administrations of ethanol (2.2 g/kg) or saline every other day for 10 days. According to their locomotor response on the last day of treatment, ethanol-treated animals were classified into two groups: sensitized or non-sensitized mice. After the treatment, mice were submitted to four challenges 48 h apart. In experiments 1 and 2, mice were challenged, respectively, with i.p. administration of opioid antagonists (naloxone or naltrexone) or an opioid agonist (morphine), followed immediately by 2.2 g/kg ethanol. In experiment 3, animals received morphine by i.c.v., followed by 2.2 g/kg of ethanol (i.p.). Pretreatment with opioid antagonists (naloxone or naltrexone) did not block the expression of ethanol sensitization; however pretreatment with morphine attenuated the increased locomotor activity after ethanol administration in sensitized mice. In experiment 4, after the ethanol or saline treatment, mice brains were processed and brain mu opioid binding was assessed by autoradiography using [3H]D-Ala2,N-mePhe4, Gly-ol5-enkephalin ([3H]DAMGO). No differences were seen between any of the groups of mice, so the agonist effect is not likely to be mediated by differences in binding to mu opioid receptors.     

Adenosine, through the A1 receptor, inhibits vesicular MHC class I cross-presentation by resting DC

Levels of the purine nucleoside adenosine (Ado) increase during conditions related to hypoxia such as inflammation, tissue damage and cancer. Ado binds to a set of G protein-linked receptors (A(1), A(2A), A(2B) and A(3)) which are widely and differentially expressed in tissues and regulate inflammatory and autoimmune responses. We have investigated the effect of the stable Ado analogue 2-Chloro-Adenosine (2-Cl-Ado) on vesicular MHC class I cross-presentation using the exogenous protein ovalbumin (OVA) and cultured mouse dendritic cells (DC) at different stages of maturation and activation. 2-Cl-Ado was found to strongly inhibit cross-presentation of OVA by resting DC (rDC) but had a much smaller effect on immature and CpG-activated DC. The effect of Ado on rDC could be fully reversed by the Ado receptor antagonist CGS 15943 and by pertussis toxin demonstrating that it was mediated by a Gi-linked Ado receptor. A(1) Ado receptor mRNA was found to be upregulated in rDC and, by using rDC from A(1), A(2A) and A(3) receptor knockout mice, this receptor was found to mediate most of the suppression. 2-Cl-Ado did not influence the cellular uptake of OVA, the cytosolic processing of the protein in rDC or the formation of intracellular MHC-I/peptide complexes in a LAMP1 positive vesicular compartment but inhibited the transport of these to the cell surface. It is concluded that 2-Cl-Ado, by acting on A(1) receptor, can suppress vesicular MHC class I cross-presentation in rDC.     

Leptin receptor signaling supports cancer cell metabolism through suppression of mitochondrial respiration in vivo

Obesity represents a risk factor for certain types of cancer. Leptin, a hormone predominantly produced by adipocytes, is elevated in the obese state. In the context of breast cancer, leptin derived from local adipocytes is present at high concentrations within the mammary gland. A direct physiological role of peripheral leptin action in the tumor microenvironment in vivo has not yet been examined. Here, we report that mice deficient in the peripheral leptin receptor, while harboring an intact central leptin signaling pathway, develop a fully mature ductal epithelium, a phenomenon not observed in db/db mice to date. In the context of the MMTV-PyMT mammary tumor model, the lack of peripheral leptin receptors attenuated tumor progression and metastasis through a reduction of the ERK1/2 and Jak2/STAT3 pathways. These are tumor cell-autonomous properties, independent of the metabolic state of the host. In the absence of leptin receptor signaling, the metabolic phenotype is less reliant on aerobic glycolysis and displays an enhanced capacity for β-oxidation, in contrast to nontransformed cells. Leptin receptor-free tumor cells display reduced STAT3 tyrosine phosphorylation on residue Y705 but have increased serine phosphorylation on residue S727, consistent with preserved mitochondrial function in the absence of the leptin receptor. Therefore, local leptin action within the mammary gland is a critical mediator, linking obesity and dysfunctional adipose tissue with aggressive tumor growth.     

Brefeldin A inhibits vesicular MHC class I processing in resting but not in CpG- and disruption-activated DC

Dendritic cells (DC) can process exogenous proteins for presentation on MHC class I (MHC-I) in a vesicular pathway (VP) that is distinct from the classical, cytosolic MHC-I pathway. Here we investigate the sensitivity of this VP to Brefeldin A (BFA), a fungal metabolite which inhibits vesicular trafficking by preventing the activation of some ADP-ribosylation factor (ARF) proteins, in both resting and differently activated DC. The VP could be directly visualized in DC by the presence of OVA-derived H-2K(b)/SIINFEKL complexes in a LAMP1 positive, but EEA negative, compartment in wt but not in cathepsin S(-/-) mice as these are unable to process OVA into the SIINFEKL peptide in endolysosomes. BFA, which binds to specific ARF-GDP-sec7 sites, both in the Golgi and in endolysosomes, was found to bind to and inhibit the VP in resting DC. If the VP was selectively activated with an immunostimulatory CpG ODN, binding to endolysosomal TLR9 receptors, or by the mere mechanical disruption of clustered DC cells, BFA no longer had this effect. The activation of the VP with both CpG and cellular disruption was found to be dependent on the MyD88 adaptor protein. We conclude that vesicular MHC-I processing in DC occurs in ARF-regulated LAMP1 positive vesicles which, as a consequence of cellular activation, no longer can bind BFA and thus become resistant to the inhibitory effect of this drug.     

Morphine, but not atropine, blocks nociceptor-driven activity in rat dorsal hippocampal neurones

Neurones in the CA1 region of the dorsal hippocampus of urethane-anaesthetized rats were recorded extracellularly with microelectrodes, and tested for their response to non-noxious and noxious peripheral stimuli. None of the cells responded to non-noxious stimuli but 91 of 216 cells were excited by noxious stimuli. Morphine (5.0 mg/kg i.v.) increased the background firing rate in 7 of 10 cells tested and blocked the response to noxious heating of the tail in all cases. Naloxone (0.5 mg/kg i.v.) reversed these effects of morphine. Atropine (0.1 plus 0.2 mg/kg i.v.), in another group of animals, produced only transient changes of an inconsistent nature in 7 cells tested.     

Measles virus-dendritic cell interaction via SLAM inhibits innate immunity: selective signaling through TLR4 but not other TLRs mediates suppression of IL-12 synthesis

Two hallmarks of measles virus (MV) infection are the ability of the virus to cause immunosuppression and the resultant enhanced susceptibility of the infected host to microbial insults. We investigated the effect of MV infection on the ability of dendritic cells (DCs) to induce IL-12 via toll-like receptor (TLR) signaling. When infected with MV, transgenic mice which expressed human SLAM receptor on their DCs were defective in the selective synthesis of IL-12 in DCs in response to stimulation of TLR4 signaling, but not to engagements of TLR2, 3, 7 or 9. MV suppressed TLR4-mediated IL-12 induction in DCs even in the presence of co-stimulation with another ligand for TLR2, 3, 7, or 9. While MV V and C proteins were not responsible for IL-12 inhibition, interaction of MV hemagglutinin with human SLAM facilitated the suppression. These results suggest that MV, by altering DC function, renders them unresponsive to secondary pathogens via TLR4.     

Nitrogen-containing bisphosphonate,YM529/ONO-5920, inhibits tumor metastasis in mouse melanoma through suppression of the Rho/ROCK pathway

The small GTPases of the Ras and Rho families are widely involved in tumorigenesis and metastasis. We recently showed that YM529/ONO-5920, a new developed bisphosphonate, inhibits the mevalonate pathway, is required for the prenylation of the small GTPases. In this study, we investigated whether YM529/ONO-5920 inhibits tumor cell migration, invasion, adhesion, and metastasis in B16BL6 cells, a mouse melanoma cell line. It was found that YM529/ONO-5920 significantly inhibited lung metastasis, cell migration, invasion, and adhesion at concentrations that did not show anti-proliferative effects on B16BL6 cells. YM529/ONO-5920 also inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs). Furthermore, YM529/ONO-5920 suppressed Rho activation, but not activation of Ras. The results indicate that YM529/ONO-5920 suppresses the Rho/ROCK pathways, thereby inhibiting B16BL6 cell migration, invasion, adhesion and metastasis. These findings suggest that YM529/ONO-5920 has potential clinical applications for the treatment of tumour cell metastasis.     

Ligation of CD5 on resting B cells,but not on resting T cells,results in apoptosis

The CD5 molecule is expressed by a B cell subset. We have demonstrated that resting B cells do not proliferate in response to CD5 ligation, whereas cells preactivated with anti-IgM and IL-2 do so. Here, we specifically studied the effects of anti-CD5 and anti-IgM on apoptosis of CD5⁺ B cells. Both ligation of CD5 or of surface IgM (sIgM) resulted in apoptosis. This started earlier following ligation of CD5 than with sIgM, and both responses were time dependent. CD5-induced apoptosis was independent of the epitope recognized or the way the antibody was presented to the B cells. CD5⁺ B cells were more sensitive to IgM-induced apoptosis than CD5⁻ B cells. Engagement of CD5 or CD3 expressed by T cells failed to induce apoptosis. Our data indicate differences in the function of CD5 molecules on tonsillar B cells, compared with blood T cells and suggest that cross-linking CD5 on B cell activates specific pathways responsible for apoptosis.     

Signaling through CD19, Fc receptors or transforming growth factor-beta: each inhibits the activation of resting human B cells differently.

To understand further the roles that negative regulatory signals may play in B cell immune responses, we compared three inhibitors of B cell proliferation: cross-linking CD19 with monoclonal antibody (mAb), signaling through Fc receptors by intact anti-mu mAb, and transforming growth factor-beta (TGF-beta). Each agent was tested for its ability to block proliferation and specific activation events induced in human tonsilar B cells activated by either cross-linking surface immunoglobulin, signaling through CD20, or direct activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate. We found that each inhibitor was functionally distinct. Both anti-CD19 mAb and anti-mu mAb inhibited anti-immunoglobulin activated cells and anti-CD20-activated cells, but neither inhibited cells activated by phorbol 12-myristate 13-acetate. TGF-beta, on the other hand, inhibited equally profoundly cells activated by each of the three regimens. These results suggest that TGF-beta blocks B cell activation at a step following the activation of PKC, whereas both signaling through CD19 and Fc receptor block early steps in the PKC activation pathway. Signaling through anti-CD19 mAb was unique in that proliferation of anti-immunoglobulin-activated cells was reduced on day 3 and then augmented subsequently. With all other inhibitory combinations the block was permanent. We conclude that each of these three inhibitors has unique important functions and therefore suggest that the effectiveness of negative signaling in B cell immune regulation will depend on the combinations of specific inhibitors modulating a specific activation program.     

Passive membrane properties,afterpotentials and repetitive firing of superior colliculus neurons studied in the anesthetized cat

Intracellular recording and staining with HRP were used to characterize cat superior colliculus neurons with identified projection into the tecto-bulbo-spinal tract (TBSNs). TBSNs are large multipolar neurons with heavy stem dendrites. First and second order dendrites bifurcate with an average branch power n of about 3/2. More peripheral branch points have n less than 1.5. Input resistances of TBSNs range from 0.9 to 4.6 M omega. Most TBSNs display 'anomalous rectification'. Based on Rall's steady-state cable equations, input resistances were calculated for 3 TBSNs labelled with HRP. Assuming a specific membrane resistance of 2,300-2,600 omega cm2 the/calculated values agree well with the experimentally determined estimates from another set of non-stained TBSNs. Membrane time constants of TBSNs range from 3.0 to 5.6 ms. The electrotonic length was calculated using the ratio tau 0/tau 1. The respective average value was 1.13. TBSNs respond to orthodromic, antidromic and direct stimulation with action potentials of 60-80 mV, composed of IS- and SD-components. The critical interval for IS-SD-invasion was on average 1.6 ms. Spike decomposition occurs usually at M-level. The postspike conductance increase underlying hyperpolarizing afterpotentials (HAP) decays exponentially, with the time constants tau F = 1.5 ms and tau S = 13 ms. The HAP was equilibrated at membrane potentials of -73 to -90 mV. When tested by antidromic stimuli at varying intervals most TBSNs show very poor "summation" of HAPS. A pronounced depolarizing hump (DD) follows antidromic action potentials. Discharging at short intervals leads to a substantial increase and prolongation of DD. This apparent DD-potentiation is interpreted as a phenomenon secondary to the reduction of hyperpolarizing currents. In response to directly injected currents, TBSNs discharge with frequencies up to 1,100 imp/s. The frequency-current curves of TBSNs are characterized by 3 ranges. The average f-i-slopes of the adapted discharge were 19.2 imp/s/nA and 56.4 imp/s/nA for the 1st and 2nd range, respectively. At intermediate current intensities (2nd range) TBSNs discharge in groups of 2 to 7 action potentials, following each other at intervals of 1.0-2.8 ms. The spike groups are separated by pauses of 3.5-6.3 ms duration. The transition from 1st (low frequency continuous) discharge range to 2nd (grouped) discharge range is related to the appearance of extra-spikes. Extra-spikes are generated from a decreased firing level, from the peak of an enhanced DD.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective destruction of medial septal cholinergic neurons attenuates pyramidal cell suppression, but not excitation in dorsal hippocampus field CA1 induced by subcutaneous injection of formalin

Using extracellular recording techniques in urethane- (1g/kg, i.p.) anaesthetized rats, we investigated the influence exercised by medial septal cholinergic neurons on dorsal hippocampus field CA1 neural responses to a hind paw injection of formalin (5%, 0.05 ml, s.c.). Cholinergic neurons of the medial septal region were destroyed by local microinjection of the immunotoxin 192 IgG-saporin. Compared to control vehicle microinjected animals, immunotoxin-treatment attenuated the amplitude, but not frequency, of CA1 theta induced by intraseptal injection of carbachol. This suggested a selective destruction of medial septal cholinergic neurons by the immunotoxin. Such destruction also abolished; (i) intraseptal carbachol-induced suppression of CA1 population spike, and (ii) stimulation-intensity dependent increase in amplitude, but not frequency, of theta evoked on electrical stimulation in the region of oral part of pontine reticular nucleus. Further, in comparison to vehicle-treated animals, selective cholinergic destruction attenuated formalin-induced; (i) theta activation, (ii) suppression of CA1 pyramidal cell population spike and dendritic field excitatory post-synaptic potential, (iii) inhibition of complex spike cell extracellular activity, and (iv) excitation and theta-rhythmicity of local putative GABAergic interneurons. However, pretreatment with the immunotoxin did not alter the strength and proportion of complex spike cells excited following injection of formalin. From these findings we suggest that medial septal cholinergic neurons mediate, at least partly, the amplitude of theta and pyramidal cell suppression via an inhibitory network involving CA1 interneurons. The data also indicates that during formalin theta, the cholinergic-mediated inhibitory processing does not modulate the strength and selectivity of complex spike cell excitation. This points to formalin-induced, non-overlapping inhibitory and excitatory processes that might have different functional relevance.     

The immunosuppressive agent FK506 inhibits in vitro expression of membrane-bound and soluble interleukin-2 receptors on resting but not on activated human lymphocytes.

FK506 is a recently introduced immunosuppressive agent synthesised by the microorganism Streptomyces tskubaensis. It has been found to be more potent than Cyclosporin A in inhibiting T cell activation. We investigated its effects on the expression of membrane bound as well as soluble interleukin-2 receptors on human lymphocytes. The membrane-bound IL-2 receptor expression was inhibited by FK506 in resting lymphocytes at a concentration of 1 pmol/l. At 10 nmol/l no further inhibition was seen. In activated lymphocytes FK506 exerted no inhibitory effect on the IL-2 receptor expression. The release of soluble IL-2 receptor showed a pronounced decline in the concentration interval between 10 pmol/l and 0.1 nmol/l. Above a concentration of 10 nmol/l, no further decrease was seen. In activated lymphocytes the expression of soluble IL-2 receptors was unaffected by FK506 incubated up to 72 h. Pretreatment of the lymphocytes with the compound did not further depress the expression of the membrane-bound or the soluble receptor. Our results also indicate that the expression of the membrane-bound receptor is more sensitive to the drug than the soluble form of the receptor.     

Reducing IRF5 expression attenuates colitis in mice,but impairs the clearance of intestinal pathogens

IRF5 genetic variants leading to decreased IRF5 expression reduce risk for ulcerative colitis. However, how IRF5 regulates intestinal inflammation and contributes to the balance between defenses against intestinal pathogens and inflammation in vivo, and the cells mediating this balance, are not known. We found that deleting IRF5 in mice led to reduced intestinal inflammation in the T cell transfer colitis model, with reduced Th1 and Th17, and increased Th2 cytokines. However, with orally-administered invasive S. Typhimurium, IRF5^−/− mice demonstrated an increased bacterial burden in the context of reduced Th1 and Th17 cytokines. IRF5 in macrophages was required for PDK1-dependent phagocytosis and for NFκB-dependent pathways mediating intracellular bacterial clearance. Despite reduced bacterial clearance pathways, in IRF5^−/− mice exposed to high levels of resident intestinal bacteria after DSS-induced injury, the lower levels of inflammatory cytokines were associated with reduced intestinal permeability, and in turn, reduced bacterial translocation and intestinal inflammation. Consistent with the myeloid cell-intrinsic roles for IRF5 in vitro, mice with IRF5 deleted from myeloid cells demonstrated outcomes similar to those observed in IRF5^−/− mice. While these data suggest that inhibition of IRF5 may be therapeutic in colitis, this needs to be balanced with the identified IRF5 role in protecting against intestinal pathogens.