IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle.

In testing the hypothesis that interleukin-4 receptor alpha-subunit (IL-4R alpha)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4R alpha blocking antibody (anti-IL-4R alpha Ab). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4R alpha Ab. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to naïve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4R alpha-coupled release and action of IL-5 in the sensitized ASM itself.

Rhinovirus elicits proasthmatic changes in airway responsiveness independently of viral infection.

BACKGROUND: Rhinovirus (RV), the principal pathogen responsible for the common cold, is importantly implicated in triggering attacks of asthma secondary to changes in airway responsiveness. OBJECTIVE: Because the airway histopathologic features of RV infection are relatively modest, we tested the hypothesis that RV can directly elicit proasthmatic-like changes in airway smooth muscle (ASM) responsiveness independently of actual viral infection and its associated cytopathic effects. METHODS: Isolated ASM tissues and cultured ASM cells were inoculated with either infectious or noninfectious (UV-irradiated) RV16 and RV2, the latter serotypes belonging to the "major" and "minor" groups of RV subtypes, respectively. ASM constrictor and relaxant responsiveness, G(i) protein expression, and proinflammatory cytokine release were subsequently compared under the different treatment conditions. RESULTS: In contrast to RV2, which had no effect, RV16 inoculation elicited enhanced ASM contractility and impaired relaxation to cholinergic and beta-adrenergic agonists, respectively, in association with increased ASM membrane G(i) protein expression and induced release of the proinflammatory cytokines IL-5 and IL-1beta. These proasthmatic-like effects were also observed in ASM exposed to UV-irradiated RV16, wherein viral replication was completely inhibited. In contrast, pretreatment of ASM with a neutralizing antibody directed against ICAM-1, the host receptor for the "major" group of RVs, completely abrogated the proasthmatic effects of RV16. CONCLUSIONS: The results demonstrate that (1) RV16 elicits proasthmatic changes in ASM responsiveness that can occur independently of actual viral infection of the ASM and (2) the effects of RV16 are attributed solely to binding of the virus to its host receptor (ICAM-1) on the ASM cell surface. Collectively, these findings support the notion that RV-induced exacerbation of wheezing in asthmatic individuals can occur even in the absence of any cytopathology associated with viral infection.

Autocrine signaling by IL-10 mediates altered responsiveness of atopic sensitized airway smooth muscle.

To elucidate the role and mechanism of action of interleukin (IL)-10 in regulating airway smooth muscle (ASM) responsiveness in the atopic asthmatic state, isolated rabbit tracheal ASM segments were passively sensitized with serum from atopic asthmatic patients or nonatopic nonasthmatic (control) subjects in both the absence and presence of an anti-IL-10 receptor blocking antibody (Ab). Relative to control ASM, atopic asthmatic serum-sensitized tissues exhibited enhanced isometric constrictor responses to administered acetylcholine and attenuated the relaxation responses to isoproterenol. These proasthmatic effects were prevented in atopic asthmatic serum-sensitized ASM that was pretreated with anti-IL-10 receptor Ab. In complementary experiments, exposure of cultured human ASM cells to atopic asthmatic serum induced upregulated expression of IL-10 mRNA. Moreover, extended studies demonstrated that 1) exogenous IL-10 administration to naive ASM elicited augmented contractility to acetylcholine and impaired relaxation to isoproterenol, 2) these effects of IL-10 were prevented by pretreating the tissues with an IL-5 receptor Ab, and 3) IL-10 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of atopic asthmatic serum-sensitized ASM is largely attributed to activation of an intrinsic T helper type 2-type autocrine mechanism involving IL-10-mediated release and the action of IL-5 in the sensitized ASM itself.

Isoforms of vascular endothelial growth factor act in a coordinate fashion To recruit and expand tumor vasculature.

Vascular endothelial growth factor (VEGF) is an essential regulator of vascularization. It is expressed as several splice variants; the major forms contain 120 amino acids, 164 amino acids, and 188 amino acids. We utilized transformed cells nullizygous for VEGF to specifically express each of these isoforms in isolation, in order to determine the role of each in tumorigenic neo-vascularization. We found that only the intermediate isoform, VEGF164, could fully rescue tumor growth; VEGF120 partially rescued tumor growth, and VEGF188 failed completely to rescue tumor expansion. Surprisingly, the vascular density of VEGF188 isoform-expressing tumors is significantly greater than that of wild-type VEGF cells and the other isoform-specific tumors. The failure of the hypervascular VEGF188-expressing tumors to grow may be due to inadequate perfusion of the massive number of microvessels in these tumors; three-dimensional imaging of the tumorigenic vasculature indicated little or no recruitment of the peripheral vasculature. This demonstrates that the VEGF isoforms perform unique functions which together enable tumorigenic vascularization.

A diagnostic service for eliciting carotid sinus hypersensitivity and vasovagal symptoms in a district general hospital.

AIM: to describe the technical, diagnostic and logistical problems encountered in a syncope and falls clinic in a district general hospital. METHODS: we have reviewed 157 consecutive patients and the problems encountered in their assessment at the clinic. RESULTS: 143 patients (91%) completed assessment, which included head-up tilt and carotid sinus massage. We reached a diagnosis in 75 of these (52%). Difficulties with continuous blood pressure monitoring caused testing to be abandoned in four cases and caused considerable delay (up to 30 min) in 45% of the rest. Eight patients (5%) refused consent for carotid sinus massage and two others had neurological sequelae. Some patients fulfilled diagnostic criteria on testing but remained asymptomatic, making an attributable diagnosis difficult. This was most noticeable with vasodepressor carotid sinus hypersensitivity. The limited facilities in a district general hospital and the time-consuming nature of the testing resulted in a considerable delay between referral and assessment (58 days +/- 25.9), which may adversely affect diagnostic yield. CONCLUSIONS: syncope clinics have a role in the assessment of elderly people with recurrent syncope and unexplained falls. Enthusiasm for this approach has to be tempered with an awareness of the limitations of the tests involved and an appreciation of the logistical problems likely to be encountered in a district hospital.

Knockout of the glutamate dehydrogenase gene in bloodstream Trypanosoma brucei in culture has no effect on editing of mitochondrial mRNAs.

Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.

Tumor-derived expression of vascular endothelial growth factor is a critical factor in tumor expansion and vascular function.

There is considerable controversy concerning the importance of tumor-derived angiogenic factors to the neovascularization of solid tumors. Tumor, endothelial, and stromal expression of vascular endothelial growth factor (VEGF) have been hypothesized to be critical for tumor angiogenesis. To determine the relative contribution of tumor versus nontransformed tissue expression of VEGF to tumor growth, we used gene targeting and cre-loxP recombination to generate embryonic stem cell lines in which VEGF can be conditionally deleted. These lines were used to derive mouse embryonic fibroblast lines with null mutations in both alleles of VEGF. Upon immortalization and H-ras transformation, we used these VEGF null fibroblasts to make fibrosarcomas in immunocompromised mice. We report that tumorigenic VEGF expression is critical for ras-mediated tumorigenesis, and the loss of tumorigenic expression causes dramatic decreases in vascular density and vascular permeability and increases in tumor cell apoptosis.

Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract.

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.

Endogenous opioids modulate fetal rabbit lung maturation.

To test the hypothesis that endogenous opioids modulate fetal lung development, separate groups of pregnant rabbits received daily injections of saline, morphine (1 mg/kg body wt), or the opioid antagonist naloxone (0.4 and 5.0 mg) for 10 days during their last trimester of pregnancy. The corresponding groups of fetuses were then delivered prematurely on day 28 of gestation (term approximately 31 days) and evaluated with respect to differences in body weight, lung weight, and the ratios of wet to dry lung weight and lung dry weight to body weight, the static inflation and deflation air and saline pressure-volume (P-V) characteristics of the lungs, and lung morphology. Mean values for body weight, lung weight, and the ratios of lung wet to dry weight and lung dry weight to body weight were not significantly different among the saline control (C), morphine (M)-, and naloxone (NLX)-treated fetuses. On the other hand, the fetal air P-V curves varied significantly (P less than 0.001), wherein the M-treated group depicted increased lung distensibility and alveolar stability on lung deflation, whereas the opposite was obtained in the NLX-treated fetuses. Moreover, morphometric analyses demonstrated that the mean alveolar air space-to-tissue ratio in lungs from M-treated fetuses were significantly greater than that observed either in C or in NLX-treated fetuses (P less than 0.05); however, the air space-to-tissue ratio did not significantly vary between the C and NLX-treated animals. These observations provide new evidence that endogenous opioids enhance fetal lung maturation.

Mechanism of substance P-induced bronchoconstriction in maturing rabbit.

The maturational effects of substance P (SP) on airway function were quantitatively assessed in 30 anesthetized tracheotomized rabbits ranging in age from 2 to 29 days. Following paralysis, during mechanical ventilation with 100% O2 in a body plethysmograph, respiratory resistance (Rrs) and dynamic compliance (Cdyn) were continuously monitored. Noncumulative systemic infusions of SP (0.001-5.0 micrograms/g) produced dose-dependent decreases in pulmonary conductance (Grs), i.e., 1/Rrs, and Cdyn. The dose of SP producing a 50% decrease in Cdyn (PD50-Cdyn) significantly increased as a function of age indicating a diminution in airway sensitivity to SP. Bilateral cervical vagotomy and ganglionic blockade with hexamethonium had no effect on the airway response to SP. On the other hand, the response was significantly reduced following atropine sulfate infusion (2 mg/kg), suggesting a peripheral cholinergic contribution located distal to the airway parasympathetic ganglia. The magnitude of this cholinergic contribution increased as a function of age. Unlike atropine, antagonists to histamine and 5-hydroxytryptamine had no effect on the airway response to SP, however, the response was inhibited following infusion of the SP antagonist, D-Pro2,D-Trp7,9-SP. These findings indicate that the airway response to SP is age-related and mediated by binding of the agonist to airway smooth muscle coupled with an accelerated release of acetylcholine at the airway neuromuscular junction.

Maturational effect of enkephalin on respiratory control in newborn rabbits.

The respiratory responses to systemic infusion of the opioid peptide, [D-Ala2, D-Leu5]enkephalin (ENK) were determined in 39 unanesthetized tracheotomized rabbits (age range 1-20 days). At all ages, ventilation (VE), measured in a body plethysmograph, was depressed after ENK infusion in association with a decrease in CO2 elimination (VCO2) and body temperature. The degree of VE depression varied inversely with increasing age and was directly related to changes in mean inspiratory flow (i.e., VT/TI) while the ratio of inspiratory to total breath duration (TI/TT) was unaltered, except in rabbits under about 1 wk of age. Maturational differences in the VE response to ENK were related to age-dependent variation in the stability of the central inspiratory activity, which was manifested as periodic breathing with apnea in rabbits under about 5 days of age. Since the initial inspiratory volume-time profile was little affected by ENK and vagal afferent influence on respiration was not diminished, the depression in VE could be explained by an inhibition of the central inspiratory "off-switch" threshold and delay in central inspiratory "on-switching." All effects of ENK were reversed by the opiate antagonist, naloxone.