Activation of C3a receptor is required in cigarette smoke-mediated emphysema.

Exposure to cigarette smoke can initiate sterile inflammatory responses in the lung and activate myeloid dendritic cells (mDCs) that induce differentiation of T helper type 1 (Th1) and Th17 cells in the emphysematous lungs. Consumption of complement proteins increases in acute inflammation, but the contribution of complement protein 3 (C3) to chronic cigarette smoke-induced immune responses in the lung is not clear. Here, we show that following chronic exposure to cigarette smoke, C3-deficient (C3(-/-)) mice develop less emphysema and have fewer CD11b(+)CD11c(+) mDCs infiltrating the lungs as compared with wild-type mice. Proteolytic cleavage of C3 by neutrophil elastase releases C3a, which in turn increases the expression of its receptor (C3aR) on lung mDCs. Mice deficient in the C3aR (C3ar(-/-)) partially phenocopy the attenuated responses to chronic smoke observed in C3(-/-) mice. Consistent with a role for C3 in emphysema, C3 and its active fragments are deposited on the lung tissue of smokers with emphysema, and smoke-exposed mice. Together, these findings suggest a critical role for C3a through autocrine/paracrine induction of C3aR in the pathogenesis of cigarette smoke-induced sterile inflammation and provide new therapeutic targets for the treatment of emphysema.

Multistrain influenza protection induced by a nanoparticulate mucosal immunotherapeutic.

All commercial influenza vaccines elicit antibody responses that protect against seasonal infection, but this approach is limited by the need for annual vaccine reformulation that precludes efficient responses against epidemic and pandemic disease. In this study we describe a novel vaccination approach in which a nanoparticulate, liposome-based agent containing short, highly conserved influenza-derived peptides is delivered to the respiratory tract to elicit potent innate and selective T cell-based adaptive immune responses. Prepared without virus-specific peptides, mucosal immunostimulatory therapeutic (MIT) provided robust, but short-lived, protection against multiple, highly lethal strains of influenza in mice of diverse genetic backgrounds. MIT prepared with three highly conserved epitopes that elicited virus-specific memory T-cell responses but not neutralizing antibodies, termed MITpep, provided equivalent, but more durable, protection relative to MIT. Alveolar macrophages were more important than dendritic cells in determining the protective efficacy of MIT, which induced both canonical and non-canonical antiviral immune pathways. Through activation of airway mucosal innate and highly specific T-cell responses, MIT and MITpep represent novel approaches to antiviral protection that offer the possibility of universal protection against epidemic and pandemic influenza.

Multiple zinc binding sites in retinal rod cGMP phosphodiesterase, PDE6alpha beta.

The photoreceptor cGMP phosphodiesterase (PDE6) plays a key role in vertebrate vision, but its enzymatic mechanism and the roles of metal ion co-factors have yet to be determined. We have determined the amount of endogenous Zn(2+) in rod PDE6 and established a requirement for tightly bound Zn(2+) in catalysis. Purified PDE6 contained 3-4-g atoms of zinc/mole, consistent with an initial content of two tightly bound Zn(2+)/catalytic subunit. PDE with only tightly bound Zn(2+) and no free metal ions was inactive, but activity was fully restored by Mg(2+), Mn(2+), Co(2+), or Zn(2+). Mn(2+), Co(2+), and Zn(2+) also induced aggregation and inactivation at higher concentrations and longer times. Removal of 93% of the tightly bound Zn(2+) by treatment with dipicolinic acid and EDTA at pH 6.0 resulted in almost complete loss of activity in the presence of Mg(2+). This activity loss was blocked almost completely by Zn(2+), less potently by Co(2+) and almost not at all by Mg(2+), Mn(2+), or Cu(2+). The lost activity was restored by the addition of Zn(2+), but Co(2+) restored only 13% as much activity, and other metals even less. Thus tightly bound Zn(2+) is required for catalysis but could also play a role in stabilizing the structure of PDE6, whereas distinct sites where Zn(2+) is rapidly exchanged are likely occupied by Mg(2+) under physiological conditions.

Distribution of camptothecin after delivery as a liposome aerosol or following intramuscular injection in mice.

PURPOSE: The plant alkaloid camptothecin (CPT) has shown significant antitumor activity against a wide variety of human tumors xenografted in nude mice. In previous studies we have found that administration of dilauroylphosphatidylcholine (DLPC) liposome aerosols containing 9-nitrocamptothecin (9-NC) inhibits the growth of human breast, colon and lung cancer xenografts. The purpose of this study was to analyze the pharmacokinetics and tissue distribution of inhaled CPT formulated in DLPC liposomes. METHODS: C57BL/6 mice with subcutaneous Lewis lung carcinoma, Swiss nu/nu mice with human lung carcinoma xenografts and BALB/c mice without tumors were used for pharmacokinetic studies of CPT administered as a liposome aerosol and BALB/c mice were given CPT intramuscularly. RESULTS: After 30 min inhalation of CPT liposome aerosol, drug was deposited in the lungs (310 ng/g) and was followed promptly by the appearance of high concentrations in the liver (192 ng/g) and with lesser amounts appearing in other organs. Drug concentration in the brain was 61 ng/g. After intramuscular injection of CPT dissolved in DMSO, drug was released from the site of injection very slowly and accumulated mainly in the liver (136 ng/g). Only trace amounts appeared in the lungs (2-4 ng/g). These results demonstrate a prompt pulmonary and later systemic distribution of CPT following liposome aerosol administration. CONCLUSIONS: The substantial concentrations of CPT in lungs and other organs following inhalation of liposome aerosol suggest the possible benefit of it and of its more active derivative, 9-NC, in the treatment of lung, liver, kidney and brain cancer in humans.

Modulation of skeletal muscle Ca2(+)-release channel activity by sphingosine.

The effect of D-erythro-C18-sphingosine (sphingosine) and related compounds on the Ca(2+)-release channel (ryanodine binding protein) was examined on rabbit skeletal muscle membranes, on the purified ryanodine binding protein, and on the channel reconstituted into planar lipid bilayers. Sphingosine inhibited [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes in a dose-dependent manner similar to published results (R. A. Sabbadini, R. Betto, A. Teresi, G. Fachechi-Cassano, and G. Salviati. J. Biol. Chem. 267: 15475-15484, 1992). The sphingolipid also inhibited [3H]ryanodine binding to the purified ryanodine binding protein. Our results demonstrate that the inhibition of [3H]ryanodine binding by sphingosine is due to an increased rate of dissociation of bound [3H]ryanodine from SR membranes and a decreased rate of association of [3H]ryanodine to the high-affinity site. Unlike other modulators of the Ca(2+)-release channel, sphingosine can remove bound [3H]ryanodine from the high-affinity site within minutes. Sphingosine increased the rate of dissociation of [3H]ryanodine bound to a solubilized proteolytic fragment derived from the carboxy terminus of the ryanodine binding protein (cleavage at Arg4475). Sphingosine also inhibited the activity of the Ca(2+)-release channel incorporated into planar lipid bilayers. Taken together, the data provide evidence for a direct effect of sphingosine on the Ca(2+)-release channel. Sphingosine is a noncompetitive inhibitor at the high-affinity ryanodine binding site, and it interacts with a site between Arg4475 and the carboxy terminus of the Ca(2+)-release channel.

Interaction between ryanodine and neomycin binding sites on Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Neomycin is a potent inhibitor of skeletal muscle sarcoplasmic reticulum (SR) calcium release. To elucidate the mechanism of inhibition, the effects of neomycin on the binding of [3H]ryanodine to the Ca2+ release channel and on its channel activity when reconstituted into planar lipid bilayer were examined. Equilibrium binding of [3H]ryanodine was partially inhibited by neomycin. Inhibition was incomplete at high neomycin concentrations, indicating noncompetitive inhibition rather than direct competitive inhibition. Neomycin and [3H]ryanodine can bind to the channel simultaneously and, if [3H]ryanodine is bound first, the addition of neomycin will slow the dissociation of [3H]ryanodine from the high affinity site. Neomycin also slows the association of [3H]ryanodine with the high affinity binding site. The neomycin binding site, therefore, appears to be distinct from the ryanodine binding site. Dissociation of [3H]ryanodine from trypsin-treated membranes or from a solubilized 14 S complex is also slowed by neomycin. This complex is composed of polypeptides derived from the carboxyl terminus of the Ca2+ release channel after Arg-4475 (Callaway, C., Seryshev, A., Wang, J. P., Slavik, K., Needleman, D. H., Cantu, C., Wu, Y., Jayaraman, T., Marks, A. R., and Hamilton, S. L. (1994) J. Biol. Chem. 269, 15876-15884). The proteolytic 14 S complex isolated with ryanodine bound produces a channel upon reconstitution into planar lipid bilayers, and its activity is inhibited by neomycin. Our data are consistent with a model in which the ryanodine binding sites, the neomycin binding sites, and the channel-forming portion of the Ca2+ release channel are located between Arg-4475 and the carboxyl terminus.

Localization of the high and low affinity [3H]ryanodine binding sites on the skeletal muscle Ca2+ release channel.

The Ca2+ release channel of skeletal muscle sarcoplasmic reticulum is modulated in a biphasic manner by the plant alkaloid ryanodine and there are two distinct binding sites on this channel for ryanodine. The Ca2+ release channel is a homotetramer with a subunit of 5037 amino acids. The ability of sarcoplasmic reticulum membranes to bind [3H]ryanodine to the high affinity site is lost upon proteolysis with trypsin. [3H]Ryanodine, however, bound before proteolysis remains bound after trypsin digestion. If the high affinity site is first occupied with [3H]ryanodine and then 100 microM ryanodine is added to occupy the low affinity sites, almost all of [3H]ryanodine bound to the high affinity site remains bound after proteolysis. Proteolysis causes the solubilized Ca2+ release channel containing bound [3H]ryanodine to undergo four discrete shifts in sedimentation (30 S-->28 S-->26 S-->19 S-->14 S). Polypeptides having apparent molecular masses of 76, 66, 56, 45, 37, and 27 kDa can be identified in the 14 S complex. The 76-, 56-, 45-, and 27-kDa polypeptides have been partially sequenced from the NH2 terminus. In addition, the 76-, 66-, and 27-kDa fragments are recognized by an antibody to the last 9 amino acids at the carboxyl terminus of the skeletal muscle ryanodine receptor and the 76-, 66-, and 37-kDa fragments are recognized by an antibody to a peptide matching the sequence 4670-4685. The 56-kDa and the 45-kDa fragments are not Ca2+ release channel fragments. Both high and low affinity ryanodine binding sites are found in the 14 S complex and are, therefore, most likely located between Arg-4475 and the carboxyl terminus.