Inhibitory effects of miRNAs in astrocytes on C6 glioma progression via connexin 43.

In many types of tumor cells, cell communication via gap junction is decreased or missing. Therefore, cancer cells acquire unique cytosolic environments that differ from those of normal cells. This study assessed the differences in microRNA (miRNA) expression between cancer and normal cells. MicroRNA microarray analysis revealed five miRNAs that were highly expressed in normal astrocytes compared with that in C6 gliomas. To determine whether these miRNAs could pass through gap junctions, connexin 43 was expressed in C6 glioma cells and co-cultured with normal astrocytes. The co-culture experiment showed the possibility that miR-152-3p and miR-143-3p propagate from normal astrocytes to C6 glioma in connexin 43-dependent and -independent manners, respectively. Moreover, we established C6 glioma cells that expressed miR-152-3p or miR-143-3p. Although the proliferation of these miRNA-expressing C6 glioma cells did not differ from that of empty vectors introduced in C6 glioma cells, cell migration and invasion were significantly decreased in C6 glioma cells expressing miR-152-3p or miR-143-3p. These results suggest the possibility that miRNA produced by normal cells attenuates tumor progression through connexin 43-dependent and -independent mechanisms.

Effect of gap junction-mediated intercellular communication on TGF-ß induced epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) is the process in which epithelial cells lose cell polarity and cell adhesion with surrounding cells to obtain migratory and invasive abilities. On the other hand, the expression of connexin is decreased or lacked in the many types of tumor cells. This study examined the effect of gap junctional intercellular communication (GJIC) on EMT induced by the transforming growth factor-ß1 (TGF-ß1). To investigate the effect of GJIC on EMT in U2OS cells, smooth muscle 22-α (sm22α) promoter-driven luciferase reporter gene was introduced into Cx43-expressing cells (U2OS-Luc Cx43) and into the control parental cell line (U2OS-Luc). TGF-ß1 induced the expression of EMT markers and the sm22α promoter activity of U2OS-Luc cells. Sm22α promoter activity of U2OS cells was neither dependent on the expression of Cx43 nor on the establishment of GJIC among U2OS cells. Furthermore, we found that the homocellular communication among tumor cells did not affected the tumor cell growth and migration. However, we revealed that tumor cell density was an important factor for tumor cells to acquire metastatic phenotype. Interestingly, the co-culture of U2OS cells with osteoblasts revealed that sm22α promoter activity was inhibited only by the GJIC established between these two cell types. These results suggest that normal osteoblast cells negatively regulate the EMT of tumor cells, at least in part. Thus, Cx43-mediated GJIC may have anti-metastatic activity in tumor cells. Our findings provide a new insight into the role of GJIC in cancer progression and metastasis and identify potential therapeutic targets for the treatment of cancer.

Surface analysis of ionic liquids with and without lithium salt using X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopy (XPS) was applied to a neat ionic liquid 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [EMI(+)][Tf(2)N(-)] and its lithium salt solution at room temperature to clarify the composition and structure of its near-surface region. Core level peaks were recorded for Li 1s, N 1s, C 1s, F 1s, O 1s, S 2s, and S 2p. Valence band XPS spectra (0-40 eV binding energy) were also studied. The XPS spectra were analyzed using DV-Xα calculations. Results show that the planar type isomer of the EMI(+) cation is dominant at the near-surface region of EMI-Tf(2)N. Results of XPS measurements show a spectrum of Li 1s in Li/EMI-Tf(2)N. The proposed models for the preferred orientation of the ions exhibit good agreement with results obtained from the DV-Xα calculations.

Human eosinophils produce and release a novel chemokine, CCL23, in vitro.

BACKGROUND: CCL23 (MPIF1/CK-BETA-8) is a novel CC chemokine that plays important roles in the inhibition of myeloid progenitor cell development, the selective recruitment of resting T lymphocytes and monocytes, and the potentiation of VEGF-induced proliferation and migration of human endothelial cells. Since eosinophils participate in the pathogenesis of airway remodeling, we examined CCL23 production and release by human eosinophils in vitro. METHODS: Using Ficoll and antibody-coated immunomagnetic beads, eosinophils and other blood cells were purified from peripheral blood samples obtained from normal subjects and mildly allergic patients. Eosinophils were cultured in the presence of 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/ml IL-5, 100 ng/ml IFN-γ, 100 ng/ml IFN-α, or immobilized secretory IgA (sIgA). Total mRNA was extracted after 6 h of culture, and mRNA expression was measured using a microarray and RT-PCR. The CCL23 concentrations in the supernatants and cell lysates after 24 and 48 h of culture were measured by ELISA. RESULTS: CCL23 mRNAs (both CK-ß8-1 and CK-ß8) were constitutively expressed in fresh eosinophils, and their expression levels were higher than in other types of blood cells. CCL23 mRNAs were significantly increased by stimulation with GM-CSF and IL-5 and slightly by IFN-α and immobilized sIgA. Fresh eosinophils contained trace amounts of CCL23 protein. CCL23 was significantly released into the supernatant when the eosinophils were stimulated with GM-CSF or IL-5 but not with IFN-γ or immobilized sIgA. CONCLUSION: Our data suggest that eosinophils produce and release CCL23 and may be involved in some in vivo physiological and pathological conditions.

Flux characteristics of cell culture medium in rectangular microchannels.

Rectangular microchannels 50 µm high and 30, 40, 50, 60, or 70 µm wide were fabricated by adjusting the width of a gap cut in a polyethylene sheet 50 µm thick and sandwiching the sheet between an acrylic plate and a glass plate. Flux in the microchannels was measured under three different inner surface conditions: uncoated, albumin-coated, and confluent growth of rat fibroblasts on the bottom of the microchannels. The normalized flux in microchannels with cultured fibroblasts or albumin coating was significantly larger than that in the uncoated channels. The experimental data for all microchannels deviated from that predicted by classical hydrodynamic theory. At small aspect ratio the flux in the microchannels was larger than that predicted theoretically, whereas it became smaller at large aspect ratio. The aspect ratio rather than Reynolds number is the correct property for predicting the variation of the normalized friction factor. We postulate that two counteracting effects, rotation of large molecules and slip velocity at the corners of the microchannels, are responsible for the deviation. From these results we conclude that albumin coating should be carried out in the same way as when fabricating our integrating cell-culture system. The outcomes of this study are not only important for the design of our culture system, but also quite informative for general microfluidics.

Analysis of signal transduction pathways involved in anti-CD30 mAb-induced human eosinophil apoptosis.

BACKGROUND: Activation of cell surface CD30 by immobilized anti-CD30 monoclonal antibodies (mAbs) induces extremely rapid and intense apoptosis in human eosinophils in vitro. This anti-CD30 mAb-induced eosinophil apoptosis was inhibited by the addition of inhibitors of p38 and ERK1/2 mitogen-activated protein kinases (MAPKs). However, the signal transduction pathways other than for MAPKs remain unclear. In the present study, we tried to clarify the molecules involved in the induction of apoptosis after cross-linking of CD30. METHODS: Purified human eosinophils were suspended in Iscove's minimal essential medium supplemented with 10% FCS and 1 ng/ml IL-5 and then cultured for 4 h in plates precoated with anti-CD30 mAb (clone Ber-H8) in the presence or absence of various signal transduction inhibitors. Eosinophil apoptosis was assessed using annexin V and flow cytometry. RESULTS: The addition of inhibitors of caspase-3, caspase-8, pan-caspases, nuclear factor kappaB and protein kinase C at reasonable concentrations did not alter anti-CD30 mAb-induced eosinophil apoptosis in vitro. However, apoptosis was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors: wortmannin at very high concentrations (>1 microM) significantly and LY294002 at a reasonable concentration partially inhibited that apoptosis. CONCLUSIONS: Anti-CD30 mAb-induced eosinophil apoptosis is likely to be mediated mainly through MAPKs and partially through PI3K, but independent of caspase activation. Downstream signaling molecules of MAPK activation in eosinophils have to be clarified in future studies.

beta2-Adrenoceptor agonists enhance cytokine-induced release of thymic stromal lymphopoietin by lung tissue cells.

BACKGROUND: Whilebeta(2)-adrenoceptor agonists (beta(2)-agonists) are widely used as bronchodilators in the treatment of asthma, there has been increasing concern that regular use of beta(2)-agonists may adversely affect the control of asthma. However, the molecular mechanisms of such undesirable effects of beta(2)-agonists are not fully understood. In this study, we examined the effects of beta(2)-agonists on cytokine-induced production of thymic stromal lymphopoietin (TSLP), an indispensable cytokine in the development of allergic diseases, by lung tissue cells. METHODS: Normal human bronchial epithelial cells (NHBE), smooth muscle cells (BSMC) and fibroblasts (NHLF) were stimulated with the IL-4 and TNF-alpha cytokines, alone and in combination, and their production of TSLP was examined by ELISA. The effects of beta(2)-agonists (salmeterol, formoterol, salbutamol), intracellular cyclic adenosine monophosphate (cAMP)-elevating agents (8-bromo-cAMP, dibutyryl cAMP, forskolin) and a corticosteroid (fluticasone) on the cytokine-induced TSLP production were examined. RESULTS: The following results were observed in all three types of lung tissue cells tested (that is, NHBE, BSMC and NHLF). Costimulation with IL-4 and TNF-alpha significantly induced TSLP production, and beta(2)-agonists further enhanced it via upregulation of intracellular cAMP. However, addition of a corticosteroid to the cytokines and beta(2)-agonist resulted in a marked decrease in TSLP production. CONCLUSIONS: beta(2)-Agonists significantly enhanced the cytokine-induced TSLP production by primary human lung tissue cells. This may be partly responsible for the undesirable clinical effects of continuous beta(2)-agonist monotherapy, and combination therapy with a corticosteroid might effectively inhibit TSLP-mediated allergic inflammation.

Amphiregulin production by human eosinophils.

BACKGROUND: Amphiregulin (AREG) plays critical roles in mammary gland development, immune responses against nematode infection, and mucous production in the lung. Since remarkable eosinophil infiltration has been shown at all of these tissue sites, we examined AREG production by human eosinophils in vitro. METHODS: Using Ficoll and antibody-coated immunomagnetic beads, eosinophils and other blood cells were purified from peripheral blood samples obtained from normal or mild allergic patients. Eosinophils were cultured in the presence of 10 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF), 10 ng/ml of IL-5, 100 ng/ml of IFN-gamma or immobilized secretory IgA. Total mRNA was extracted after 6 h of culture, and mRNA expression was measured using a microarray and RT-PCR. The AREG concentration in the supernatant and cell lysate after 48 h of culture was measured using ELISA. RESULTS: AREG mRNA was constitutively expressed in fresh eosinophils, and the expression level in the eosinophils was higher than that in other types of blood cells. AREG mRNA increased significantly upon stimulation with GM-CSF and IL-5 but not with IFN-gamma. AREG mRNA expression in GM-CSF-stimulated eosinophils was enhanced by dexamethasone. Fresh eosinophils did not contain AREG protein. AREG was significantly released into the supernatant when the eosinophils were stimulated with GM-CSF, but not with IFN-gamma or immobilized secretory IgA. CONCLUSION: Our data suggested that eosinophils produce and release AREG, and participate in some physiological and pathological conditions in vivo.

Raman spectroscopic study on alkaline metal ion solvation in 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide ionic liquid.

The Raman spectra for 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)amide [BMI][TFSA] containing alkaline metal salts of TFSA(-), MTFSA (M = Li, Na, K and Cs), were recorded in the frequency range of 200-1800 cm(-1), with varying salt concentrations at 298 K. With Li(+) and Na(+) ions, at the frequency range of 730-760 cm(-1), new Raman bands ascribable to the anion bound to the ions appeared at higher frequency relative to that found in the neat ionic liquid. On the other hand, with K(+) and Cs(+) ions, single Raman bands were solely observed. According to the difference Raman spectra for the ionic liquids containing K(+) and Cs(+), evaluated by subtracting Raman spectra for the neat ionic liquid, it turned out that two-state approximation, i.e., bulk TFSA(-) and TFSA(-) bound to K(+) and Cs(+) ions, could hold, as Li(+) and Na(+) ions. By careful analyses of Raman band intensity arising from bulk TFSA(-) as a function of the salt concentration, the solvation numbers for the respective ions were successfully evaluated to be 1.95 for Li(+), 2.88 for Na(+), 3.2 for K(+) and 3.9 for Cs(+), respectively. By taking into account that TFSA(-) acts as a bidentate ligand, the atomic coordination numbers are proposed to be 4, 6, 6 and 8 for Li(+), Na(+), K(+) and Cs(+), respectively. Raman shifts for the TFSA(-) bound to the metal ions relative to that of the bulk TFSA(-) were plotted against the ionic radii for the solvated alkaline metal ions estimated via Shannon's ionic radii, to yield a straight line with a slope of almost unity, suggesting that the electrostatic interaction predominantly operates in the ion-ion interaction between the alkaline metal ions and TFSA(-), as expected. Moreover, the Raman spectra in the frequency range of 370-450 cm(-1) strongly depend on the alkaline metal ions, indicating that cis TFSA(-) is favored in the first solvation sphere of the Li(+) ion of a relatively small ionic radius, and that such a preferred conformational isomerism of TFSA(-) diminishes with an increase of the ionic radii of the central metal ions.

Potential energy landscape of bis(fluorosulfonyl)amide.

The conformational landscape of the bis(fluorosulfonyl)amide, [FSI]-, anion was analyzed using data obtained from Raman spectroscopy, molecular dynamics (MD), and ab initio studies. The plotting of three-dimensional potential energy surfaces and the corresponding MD simulation conformer-population histograms show the existence of two stable isomers, C2 (trans) and C1 (cis) conformers, and confirm the nature of the anion as a flexible molecule capable of interconversion between conformers in the liquid state. In ionic liquids, the two [FSI]- conformers coexist in equilibrium, a result confirmed by the Raman data. The implications of the conformational behavior of the ion [FSI]- are discussed in terms of the solvation properties of the corresponding ionic liquids.

Liquid structure of room-temperature ionic liquid, 1-Ethyl-3-methylimidazolium bis-(trifluoromethanesulfonyl) imide.

The liquid structure of 1-ethyl-3-methylimidazolium bis-(trifluoromethanesulfonyl) imide (EMI(+)TFSI(-)) has been studied by means of large-angle X-ray scattering (LAXS), (1)H, (13)C, and (19)F NMR, and molecular dynamics (MD) simulations. LAXS measurements show that the ionic liquid is highly structured with intermolecular interactions at around 6, 9, and 15 A. The intermolecular interactions at around 6, 9, and 15 A are ascribed, on the basis of the MD simulation, to the nearest neighbor EMI(+)...TFSI(-) interaction, the EMI(+)...EMI(+) and TFSI(-)...TFSI(-) interactions, and the second neighbor EMI+...TFSI(-) interaction, respectively. The ionic liquid involves two conformers, C(1) (cis) and C(2) (trans), for TFSI(-), and two conformers, planar cis and nonplanar staggered, for EMI(+), and thus the system involves four types of the EMI(+)...TFSI(-) interactions in the liquid state by taking into account the conformers. However, the EMI(+)...TFSI(-) interaction is not largely different for all combinations of the conformers. The same applies alsoto the EMI(+)...EMI(+) and TFSI(-)...TFSI(-) interactions. It is suggested from the 13C NMR that the imidazolium C(2) proton of EMI(+) strongly interacts with the O atom of the -SO(2)(CF(3)) group of TFSI(-). The interaction is not ascribed to hydrogen-bonding, according to the MD simulation. It is shown that the liquid structure is significantly different from the layered crystal structure that involves only the nonplanar staggered EMI(+) and C(1) TFSI(-) conformers.

A tale of two ions: the conformational landscapes of bis(trifluoromethanesulfonyl)amide and N,N-dialkylpyrrolidinium.

The conformational landscapes of two commonly used ionic liquid ions, the anion bis(trifluoromethanesulfonyl)amide (Ntf2) and the cations N-propyl- and N-butyl-N-methylpyrrolidinium, were investigated using data obtained from Raman spectroscopy, molecular dynamics, and ab initio techniques. In the case of Ntf2, the plotting of three-dimensional potential energy surfaces (PES) and the corresponding molecular dynamics (MD) simulations confirmed the existence of two stable isomers (each existing as a pair of enantiomers) and evidenced the nature of the anion as a flexible, albeit hindered, molecule capable of interconversion between conformers in the liquid state, a result confirmed by the Raman data. In the case of the N,N-dialkylpyrrolidinium cations, the PES show a much more limited conformational behavior of the pyrrolidinium ring (pseudorotation). Nevertheless, such pseudorotation produces two stable isomers with the propyl and butyl side chains in completely different positions (axial-envelope and equatorial-envelope conformations). This result was also confirmed by Raman spectra analyses and MD simulations in the liquid phase. The implications of the conformational behavior of the two types of ions are discussed in terms of the solvation properties of the corresponding ionic liquids.

Th1/Th2 cytokines reciprocally regulate in vitro pulmonary angiogenesis via CXC chemokine synthesis.

Hypervascularity is known as an important element of airway remodeling in bronchial asthma. However, it remains obscure how allergic inflammation relates to angiogenesis in the lung. In this study, we examined the in vitro effects of inflammatory cytokines on endothelial cell functions, particularly angiogenesis. Human microvascular endothelial cells from normal lung (HMVEC-Ls) were cultured with TNF-alpha, IFN-gamma, IL-4, a combination of TNF-alpha and IFN-gamma, or a combination of TNF-alpha and IL-4, and the cell proliferation and tube-forming activities were evaluated. IL-4 slightly enhanced the proliferation of HMVEC-Ls in the presence of vascular endothelial growth factor (VEGF), whereas TNF-alpha and IFN-gamma tended to inhibit it. Synergistic inhibition was observed when TNF-alpha and IFN-gamma were simultaneously added to the culture medium. The combination of IL-4 and TNF-alpha markedly promoted tube formation by HMVEC-Ls, even in the absence of VEGF. The IL-4 and TNF-alpha combination induced autocrine production of CXCR2 chemokines, which are known to have angiogenic activity, whereas the production of angiostatic CXCR3 chemokines was dramatically up-regulated when TNF-alpha and IFN-gamma were present. The marked IL-4- and TNF-alpha-induced tube formation was inhibited by a selective CXCR2 antagonist. These results suggest that, in the presence of TNF-alpha, IL-4 and IFN-gamma reciprocally regulate tube formation by HMVEC-Ls through autocrine synthesis of CXCR2 and CXCR3 chemokines, respectively. Of note, the CXCR2 chemokine-induced tube formation was independent of VEGF. Therefore, CXCR2 chemokines may represent potential therapeutic targets for bronchial asthma.

Anion conformation of low-viscosity room-temperature ionic liquid 1-ethyl-3-methylimidazolium bis(fluorosulfonyl) imide.

Anion conformation of a low-viscosity room-temperature ionic liquid 1-ethyl-3-methylimidazolium bis(fluorosulfonyl) imide (EMI+FSI-) has been studied by Raman spectra and theoretical DFT calculations. Three strong Raman bands were found at 293, 328, and 360 cm(-1), which are ascribed to the FSI- ion. These Raman bands show significant temperature dependence, implying that two FSI- conformers coexist in equilibrium. This is supported by theoretical calculations that the FSI- ion is present as either C2 (trans) or C1 (cis) conformer; the former gives the global minimum, and the latter has a higher SCF energy of about 4 kJ mol(-1). Full geometry optimizations followed by normal frequency analyses show that the observed bands at 293, 328, and 360 cm(-1) are ascribed to the C2 conformer. The corresponding vibrations at 305, 320, and 353 cm(-1) were extracted according to deconvolution of the observed Raman bands in the range280-400 cm(-1 )and are ascribed to the C1 conformer. The enthalpy DeltaH degrees of conformational change from C2 to C1 was experimentally evaluated to be ca. 4.5 kJ mol(-1), which is in good agreement with the predicted value by theoretical calculations. The bis(trifluoromethanesulfonyl) imide anion (TFSI-) shows a conformational equilibrium between C1 and C2 analogues (DeltaH degrees = 3.5 kJ mol(-1)). However, the profile of the potential energy surface of the conformational change for FSI- (the F-S-N-S dihedral angle) is significantly different from that for TFSI- (the C-S-N-S dihedral angle).

Lithium ion solvation in room-temperature ionic liquids involving bis(trifluoromethanesulfonyl) imide anion studied by Raman spectroscopy and DFT calculations.

The solvation structure of the lithium ion in room-temperature ionic liquids 1-ethyl-3-methylimidazolium bis(trifluoromethanesulfonyl) imide (EMI(+)TFSI(-)) and N-butyl-N-methylpyrrolidinium bis(trifluoromethanesulfonyl) imide (BMP(+0TFSI(-)) has been studied by Raman spectroscopy and DFT calculations. Raman spectra of EMI(+)TFSI(-) and BMP(+)TFSI(-) containing Li(+)TFSI(-) over the range 0.144-0.589 and 0.076-0.633 mol dm(-3), respectively, were measured at 298 K. A strong 744 cm-1 band of the free TFSI(-) ion in the bulk weakens with increasing concentration of the lithium ion, and it revealed by analyzing the intensity decrease that the two TFSI(-) ions bind to the metal ion. The lithium ion may be four-coordinated through the O atoms of two bidentate TFSI(-) ions. It has been established in our previous work that the TFSI(-) ion involves two conformers of C(1) (cis) and C(2) (trans) symmetries in equilibrium, and the dipole moment of the C(1) conformer is significantly larger than that of the C(2) conformer. On the basis of these facts, the geometries and SCF energies of possible solvate ion clusters [Li(C(1)-TFSI(-))(2)](-), [Li(C(1)-TFSI(-))(C(2)-TFSI(-))](-), and [Li(C(2)-TFSI(-))(2)](-) were examined using the theoretical DFT calculations. It is concluded that the C(1) conformer is more preferred to the C(2) conformer in the vicinity of the lithium ion.

Rapid and strong induction of apoptosis in human eosinophils by anti-CD30 mAb-coated microspheres and phagocytosis by macrophages.

BACKGROUND: Eosinophils represent a potential therapeutic target in allergic diseases. We previously reported that two clones of anti-CD30 mAbs (HRS-4 and Ber-H8) induced extremely rapid and intense apoptosis in human eosinophils in vitro, but only when the mAbs were immobilized on plates [Matsumoto K, J Immunol 2004;172:2186]. As the initial step towards clinical application of these anti-CD30 mAbs in the treatment of allergic diseases, we made an attempt to clarify two issues; first, whether or not anti-CD30 mAb-coated microspheres can efficiently induce apoptosis in human eosinophils, and second, whether or not these apoptotic eosinophils can be phagocytosed by macrophages without the release of granular proteins. METHODS: Purified human eosinophils were treated with anti-CD30 mAb-coated polystyrene microspheres (diameter, 1.44 mum). Apoptosis was determined by annexin V-binding. For the phagocytosis assay, eosinophils were co-cultured with monocyte-derived human macrophages or PMA-pretreated U-937 cells. Phagocytosis was determined by light microscopy and by the eosinophil-derived neurotoxin (EDN) concentration in the supernatant. RESULTS: Anti-CD30 mAb-coated, but not control IgG1-coated microspheres significantly reduced eosinophil survival in a dose-dependent manner. Marked phagocytosis of the apoptotic eosinophils by macrophages was also observed when the eosinophils were treated with anti-CD30 mAb-coated microspheres. The apoptotic eosinophils released large amounts of EDN in the absence of macrophages; however, the EDN levels were significantly decreased when the eosinophils were co-cultured with macrophages. CONCLUSIONS: Anti-CD30 mAb-coated microspheres are capable of inducing rapid and strong apoptosis in human eosinophils. Furthermore, the apoptotic eosinophils were also phagocytosed by macrophages with minimal release of the granular proteins.

Solvation structure of Li+ in concentrated LiPF6-propylene carbonate solutions.

Time-of-flight neutron diffraction measurements were carried out for 6Li/7Li isotopically substituted 10 mol % LiPF6-propylene carbonate-d6 (PC-d6) solutions, in order to obtain structural information on the first solvation shell of Li+. Structural parameters concerning the nearest neighbor Li+...PC and Li+...PF6- interactions were determined through least-squares fitting analysis of the observed difference function, DeltaLi(Q). It has been revealed that the first solvation shell of Li+ consists in average of 4.5(1) PC molecules with an intermolecular Li+...O(PC) distance of 2.04(1) A. The angle Li+...O=C bond angle has been determined to be 138(2) degrees.

CpG oligodeoxynucleotide prolongs eosinophil survival through activation of contaminating B cells and plasmacytoid dendritic cells in vitro.

BACKGROUND: In our previous study, oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs) significantly prolonged eosinophil survival without inducing active release of eosinophil-derived neurotoxin or interleukin 8. In addition, this survival-promoting activity was nuclear factor-kappaB dependent. However, some eosinophil preparations from different donors hardly responded to CpG ODNs at all. To clarify why CpG ODN-induced nuclear factor-kB activation in eosinophils does not cause eosinophil-derived neurotoxin or interleukin 8 release and why the survival-promoting activity of CpG ODNs was not found in some eosinophil preparations, we determined the effect of extensive removal of contaminating B cells and plasmacytoid dendritic cells from human eosinophil preparations. METHODS: Eosinophils were purified from the peripheral blood of healthy or slightly allergic donors by gradient sedimentation and negative selection with anti-CD16 alone or a combination of anti-CD16, anti-CD19 and anti-blood dendritic cell antigen 4 (BDCA4) immunomagnetic beads. Eosinophil survival was measured with FITC-conjugated annexin V and propidium iodide by FACS after incubation with synthetic CpG 2006(CpG-B), CpG 2216 (CpG-A) or their GpC control ODNs for 24 h. RESULTS: The addition of anti-CD19 and anti-BDCA4 immunomagnetic beads reduced the number of contaminating CD19+ cells and CD123+ BDCA2+ cells in eosinophil preparations. CpG 2006 and CpG 2216, but not their GpC control ODNs, significantly prolonged survival of eosinophils purified with anti-CD16 immunomagnetic beads alone but not eosinophils purified with a combination of anti-CD16, anti-CD19 and anti-BDCA4 beads. CONCLUSIONS: These results strongly suggest that contaminating B cells or plasmacytoid dendritic cells in eosinophil preparations critically regulate CpG ODN-mediated prolongation of eosinophil survival and that CpG ODNs do not activate eosinophils directly.

Extremely rapid and intense induction of apoptosis in human eosinophils by anti-CD30 antibody treatment in vitro.

Apoptosis is an important cellular mechanism for controlling cell viability and proliferation. With respect to eosinophils, cytokines prolong their survival, whereas corticosteroids reduce their survival in vitro. CD30, a member of the TNFR family, is expressed on the surface of many cell types, including Hodgkin's lymphoma cells. CD30 is capable of inducing apoptosis after Ab treatment in some cell lines. To determine whether this surface structure is involved in apoptosis of human eosinophils, we examined its expression and the effect of anti-CD30 Ab treatment on the viability of eosinophils. Purified human eosinophils expressed low, but consistently detectable, levels of CD30. Immobilized, but not soluble, forms of anti-CD30 Abs (HRS-4 and Ber-H8) or recombinant mouse CD30 ligand exhibited an extremely rapid and intense survival-reducing effect on the eosinophils in the presence of exogenous IL-5; this effect was both concentration and time dependent. Furthermore, high concentrations of IL-5 could not reverse the reduced survival rates. After treatment with anti-CD30 Ab, gel electrophoresis of DNA extracted from the eosinophils demonstrated changes consistent with apoptosis. The immobilized F(ab')(2) of the anti-CD30 Ab failed to induce eosinophil apoptosis. The addition of anti-CD18 Ab also completely abrogated the induction of eosinophil apoptosis. Further examination using specific signal transduction inhibitors suggested the involvement of p38, mitogen-activated protein kinase kinase 1/2, and specific tyrosine kinase, but not NF-kappaB, in the induction of CD30-mediated eosinophil apoptosis. These data demonstrate that CD30 can modify eosinophil survival by causing an extremely rapid and intense induction of apoptosis through a tightly regulated intracellular signaling pathway.