Interference of pain control employing opioids in in vivo immunological experiments

Pain control (PC) in laboratory animals is supported by ethical as well as methodological considerations, aimed at preventing an interfering reduction in food and water intake and normalizing stress hormone levels. However, little is known about the immunomodulatory attributes of analgesics, which putatively prevents the routine implementation of PC in immunological research. In an established murine model of endotoxemia we investigated the immunomodulatory properties of common clinical analgesics (the opioids fentanyl and buprenorphine). Additionally, a literature study was conducted to investigate the frequency of PC in laboratory animals used for immunological experimentation. In line with various reports, we observed interactions between the opioid analgesics and the immune system that altered the outcome of performed in vivo immunological experiments. Of 100 evaluated publications, none mentioned the use of PC, indicating its uncommon implementation. In conclusion, more studies on the interactions between the immune system and analgesics are needed to establish better criteria for adequate implementation. Finally, we propose that methodological sections in scientific journals should clearly document whether or not PC was employed. If PC is not used, the reason for not using it should be stated.     

Biochemical aspects of the visual process,XXXV. Calcium binding by cattle rod outer segment membranes studied by means of equilibrium dialysis

The calcium binding capacity of cattle rod outer segment membranes has been studied by means of an equilibrium dialysis technique. The binding is not affected by prior lyophilization of the membranes or by the presence of ionophore A23187, indicating that only passive binding to membranes is involved without active translocation.The amount of calcium bound to the membranes is influenced by the ionic composition of the medium. Both Na⁺ and K⁺ decrease binding to about the same degree, but the size of the effects suggests a rather high specificity of the calcium binding sites on the membrane.From Scatchard plots for the amount of calcium bound as a function of the free calcium concentration, it appears that two types of binding sites exist: high affinity sites which can accommodate 5 nmol calcium per mg protein (0.3 mol. calcium/mol rhodopsin) and low affinity sites which can accommodate 195 nmol calcium per mg protein (13 mol calcium/mol rhodopsin). Depending on the medium composition, the high affinity sites show dissociation constants between 8 and 40 μM, and the low affinity sites between 0.3 and 1.6 mM.Illuminated rod outer segment membranes show a slight decrease of calcium binding as compared to dark-kept membranes, but the effect is independent of the amount of calcium bound and does not appear to be significant.From these findings and the assumption of a free calcium concentration of approx. 1 μM in the extrasaccular space in rod outer segments in vivo, it is concluded that mere passive binding to the rod sac membranes must be insufficient to explain the high calcium contents in rod outer segments.     

Requirement for CD154 in the progression of atherosclerosis.

Atherosclerosis is a systemic disease of the large arteries, and activation of inflammatory pathways is important in its pathogenesis. Increasing evidence supports the importance of CD40-CD154 interactions in atherosclerosis, interactions originally known to be essential in major immune reactions and autoimmune diseases. CD40 is present on atheroma-derived cells in vitro and in human atheromata in situ. Ligation of CD40 on atheroma-associated cells in vitro activates the production of chemokines, cytokines, matrix metalloproteinases, adhesion molecules and tissue factor, substances responsible for lesion progression and plaque destabilization. Administration of antibody against CD154 to low-density lipoprotein receptor-deficient mice has been shown to reduce atherosclerosis and decrease T-lymphocyte and macrophage content; however, only initial lesions were studied. Here, we determined the effect of genetic disruption of CD154 in ApoE-/- mice in both initial and advanced atherosclerotic lesions. Plaque area was reduced 550%. In contrast to previous reports, initial lesion development was not affected. Advanced plaques in CD154-/-ApoE-/- mice had a less-lipid-containing, collagen-rich, stable plaque phenotype, with a reduced T-lymphocyte/macrophage content. These data indicate that CD40-CD154 signaling is important in late atherosclerotic changes, such as lipid core formation and plaque destabilization.     

Tumor necrosis factor-alpha regulates the expression of inducible costimulator receptor ligand on CD34(+) progenitor cells during differentiation into antigen presenting cells

The inducible costimulator receptor (ICOS) is a third member of the CD28 receptor family that regulates T cell activation and function. ICOS binds to a newly identified ligand on antigen presenting cells different from the CD152 ligands CD80 and CD86. We used soluble ICOSIg and a newly developed murine anti-human ICOS ligand (ICOSL) monoclonal antibody to further characterize the ICOSL during ontogeny of antigen presenting cells. In a previous study, we found that ICOSL is expressed on monocytes, dendritic cells, and B cells. To define when ICOSL is first expressed on myeloid antigen presenting cells, we examined ICOSL expression on CD34(+) cells in bone marrow. We found that CD34(bright) cells regardless of their myeloid commitment were ICOSL(-), whereas ICOSL was first expressed when CD34 expression diminished and the myeloid marker CD33 appeared. However, acute myeloid leukemia cells were ICOSL-negative, whereas among B-cell malignancies only some cases of the most mature tumors such as prolymphocytic leukemia and hairy cell leukemia were positive. Next, we investigated purified CD34(+) hematopoietic progenitor cells that did not constitutively express ICOSL but were induced to express ICOSL within 12 h after granulocyte/macrophage colony-stimulating factor/tumor necrosis factor alpha (TNF-alpha) stimulation. Interestingly, ICOSL was induced prior to CD80/CD86 induction on CD34(+) cells so that ICOSL was expressed in the absence of CD80/CD86. This suggests that ICOSL is an early differentiation marker along the monocytic/dendritic maturation pathway. Induction of ICOSL was dependent on TNF-alpha and was regulated via NF-kappa B as revealed by use of inhibitors specific for I kappa B alpha phosphorylation such as BAY 11-7082 and BAY 11-7085. The antigen presenting capacity of TNF-alpha stimulated CD34(+) cells was strongly inhibited by ICOSIg fusion proteins or by NF-kappa B inhibition. Thus, TNF-alpha-induced ICOSL expression seemed to be functionally important for the costimulatory capacity of CD34(+) hematopoietic progenitor cells.     

Biochemical aspects of the visual process. XXXVI. Calcium accumulation in cattle rod outer segments: Evidence for a calcium-sodium exchange carrier in the rod sac membrane

Accumulation of calcium has been studied in bovine rod outer segments (rods), isolated by sucrose density gradient centrifugation. Calcium-depleted rods are obtained by having ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA) present during isolation.Rods thus isolated have a leaky plasma membrane, as shown by the effects of ionophore A23187 and by their light-induced phosphorylation behaviour. The accumulation of ⁴⁵Ca, determined by incubation followed by a single fast washing-filtration procedure, thus represents translocation across the rod sac membrane.Accumulation in non-depleted rods is independent of the external calcium level and of ATP, suggesting exchange of ⁴⁰Ca by ⁴⁵Ca. In depleted rods in the presence of ATP there is net uptake, sigmoidally increasing with the external calcium concentration to the level attained in non-depleted rods. This net uptake is abolished by omission of ATP, its replacement by β,γ-methylene ATP and lowering the temperature to 0° C, suggesting involvement of enzymatic hydrolysis of ATP.Replacement of KCl by NaCl in the medium causes marked inhibition of ⁴⁵Ca uptake, both net uptake and exchange. Oligomycin, ruthenium red, lanthanum and ouabain do not inhibit accumulation.Efflux of ⁴⁵Ca from pre-loaded rods is slow in a KCl medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～30}\text{min}$ at 25° C), but is greatly accelerated by addition of NaCl or Ca²⁺ ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{10}\text{s}$ at 25°C).It is concluded that the rod sac membrane contains a carrier system, which is sensitive towards Ca²⁺ and Na⁺ and which requires ATP for net uptake of Ca²⁺ but not for exchange transport of Ca²⁺ with Ca²⁺ or Na⁺.     

Endovascular renal sympathetic denervation to improve heart failure with reduced ejection fraction: the IMPROVE-HF-I study

Netherlands Heart Journal - The aim of the present study was to assess the safety and efficacy of renal sympathetic denervation (RDN) in patients with heart failure with reduced ejection fraction...     

Real-time imaging of apoptotic cell-membrane changes at the single-cell level in the beating murine heart.

We report a novel real-time imaging model to visualize apoptotic membrane changes of single cardiomyocytes in the injured heart of the living mouse, using fluorescent labeled annexin-V. Annexin-V binds to externalized phosphatidylserine (PS) of cells undergoing programmed cell death. With high-magnification (x100-160) real-time imaging, we visualized the binding of annexin-V to single cardiomyocytes. Kinetic studies at the single-cell level revealed that cardiomyocytes started to bind annexin-V within minutes after reperfusion, following an ischemic period of 30 minutes. The amount of bound annexin-V increased rapidly and reached a maximum within 20-25 minutes. Caspase inhibitors decreased the number of annexin-V-positive cardiomyocytes and slowed down the rate of PS exposure of cardiomyocytes that still bound annexin-V. This technology to study cell biology in the natural environment will enhance knowledge of intracellular signaling pathways relevant for cell-death regulation and strategies to manipulate these pathways for therapeutic effect.     

Coronary physiology assessment in a cardiac transplant patient

Netherlands Heart Journal -