IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12ß2, gp130:gp130, IL-12ß2:IL-12ß2, and IL-12ß2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rß1 and IL-23R/IL-12Rß2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

Detection of nosocomial Clostridium difficile infections with toxigenic strains despite negative toxin A and B testing on stool samples.

A two-step diagnostic algorithm is recommended to detect Clostridium difficile infections; however, samples are regularly found that are glutamate dehydrogenase (GDH) positive but stool toxin negative. In the present single-centre prospective study we focused on these 'difficult-to-interpret' samples and characterized them by anaerobic culture, toxigenic culture, slpA sequence typing and multiplex PCR (GenoType CDiff). The majority of stool toxin A and B-negative samples have been caused by toxigenic strains including ribotype 027. The multiplex PCR was faster and more sensitive compared with culture and allowed preliminary identification of hypervirulent strains in stool samples on the same day.

G proteins in Aplysia: biochemical characterization and regional and subcellular distribution.

We studied G proteins and regulation of adenylate cyclase in nervous tissue and muscle of Aplysia using bacterial toxin-catalyzed ADP-ribosylation. We identified Gs alpha, a Mr 45,000 cholera toxin substrate, Go alpha, a Mr 40,000 pertussis toxin substrate, and G beta (Mr 37,000) by Western blot analysis with antisera specific for bovine brain G protein subunits. Partial proteolysis suggests that the neuronal pertussis toxin substrates are heterogeneous. The concentration of these substrates in membranes from Aplysia ganglia is similar to that of rat, squid and Helix; in Aplysia nervous tissue, G protein subunits are most enriched in synaptosomes and neuropil. The stimulation of adenylate cyclase by serotonin (5-HT), low concentrations of GTP-gamma-S, and cholera toxin, and the inhibition by high concentrations of GTP-gamma-S that is blocked by pertussis toxin indicate that both a Gs and a Gi protein regulate the Aplysia enzyme. These results support the idea that G proteins in Aplysia are important in regulating synaptic function.

Chronic progredient diffuse alveolar damage probably related to exposure to herbicides.

Clinical history and wedge biopsy specimen findings of a Vietnam veteran suffering from progressive severe tissue damage of lung are presented. The patient served as a soldier in defoliated areas for 2 years and developed severe chest pain and dyspnoea with chronic postnasal dripping, maxillary sinusitis and allergic asthmoid bronchitis with pronounced obstructions and eosinophilia. Recurrent onsets of symptoms over a period of 10 years led to wedge biopsies of the left upper lobe, right lower lobe and mediastinal lymph node. Histology is consistent with chronic, slightly progressive diffuse alveolar damage including moderate interstitial fibrosis. Total destruction of mediastinal lymph node with deposits of amorphous material and foreign body giant cells were noted. Histology findings and clinical course favor hypersensitivity reaction of lung and congestion of exogeneous material probably related to exposure to herbicides.

The use of [125I]iodocyanopindolol as a specific probe for beta-adrenergic receptors in differentiating cultured rat skeletal muscle.

In order to examine more precisely the role of beta-adrenergic receptors in the process of differentiation we used the new radioligand iodocyanopindolol ([125I]ICYP), which we found to be a very useful probe to identify beta receptors. Binding characteristics conformed to those expected for a physiologically relevant beta receptor. L6E9 cells grown in horse serum, which allows differentiation, exhibit increased beta receptor density in intact cells as a function of age. In contrast, cells grown in fetal calf serum, which does not allow differentiation, exhibit constant beta receptor density. In broken cells, however, both differentiating and non-differentiating cells show an increase in beta receptors. These results suggest that the process of differentiation is associated with an unmasking of beta receptors which are increasing but cryptic in undifferentiated cells.

Inhibition of beta-adrenergic responsiveness in muscle cell cultures by dexamethasone.

Cultures of two myogenic cell lines, L6E9 and L8, were grown in the absence or presence of dexamethasone. Dexamethasone (1 microM) completely inhibited the formation of myotubes. Partial inhibition (20-40%) was obtained at a concentration as low as 1 nM. Dexamethasone also inhibited beta-adrenergic responsiveness, as noted by decreases in isoproterenol-stimulated adenylate cyclase activity and cAMP accumulation. These effects were both dose and time dependent. In the presence of dexamethasone, the number of beta-adrenergic receptors, as assessed by [125I]iodohydroxybenzylpindolol binding, decreased coordinately with the decrease in cAMP. A high affinity, limited capacity cytosolic binding site for [3H]triamcinolone acetonide observed under control conditions (Kd = 0.7 nM; maximum binding, 2.7 pmol/mg protein) increased in number as a function of developmental state. These data indicate that glucocorticoids inhibit myogenesis and beta-adrenergic responsiveness in vitro.

Glucocorticoids enhance glucose uptake and affect differentiation and beta-adrenergic responsiveness in muscle cell cultures.

The role of glucocorticoids (GLC) in liver glycogen metabolism is well characterized; its role in peripheral tissues is not as well understood (Baxter, 1976). GLC administration in vivo is associated with hyperglycemia, but it is not clear whether decreased glucose uptake in a peripheral tissue like muscle accounts, in part, for the effect. We investigated the relationship of glucose uptake to beta-adrenergic responsiveness in muscle cell cultures exposed to GLC. Under these conditions GLC and other serum factors are present in at least a tenfold dilution relative to in vivo conditions. We observed that the GLC dexamethasone (DEX) induces a significantly enhanced Vmax for deoxyglucose uptake in the rat muscle cell lines L8 (200--400%) and L6E9 (50--100%). DEX inhibits cell fusion and promotes epithelioid morphology within the effective dose range (L8 greater than L6E9). Growth is slightly enhanced (10--20%) at 0.1--1.0 microM. In these cells DEX also inhibits intracellular beta-adrenergic-sensitive cyclic AMP accumulation and reduces basal, catecholamine-sensitive and fluoride-sensitive adenylate cyclase in cell homogenates. The effects of DEX on deoxyglucose uptake and beta-adrenergic responsiveness are both dose (1 nM--0.1 nM) and time (1--3 days) dependent, and reversible. The degree of inhibition of the beta-adrenergic system seems to be directly related to the degree of enhancement of deoxyglucose uptake. These observations suggest that the action of DEX on muscle cell glucose uptake is related to its effect on the beta-adrenergic system.

Functional role of serotonergic neuromodulation in Aplysia.

The serotonergic metacerebral cell (MCC) of the mollusk Aplysia produces slow synaptic potentials in motor neurons of the buccal muscle, and increases the rate of ongoing rhythmic burst output of the buccal ganglion. In addition, the MCC acts peripherally to enhance the strength of buccal muscle contractions that are produced by firing of motor neurons. The potentiation of contraction is not associated with any detectable changes of resting membrane potential of muscle cells. Although MCC activity produces a small enhancement of excitatory junctional potentials, several experiments clearly indicate that the MCC has a direct potentiating effect on excitation-contraction coupling. The data suggest that potentiation of contraction might be mediated by cAMP. For example, activity of the MCC enchances the rate of accumulation of cAMP in buccal muscle, application of phosphodiesterase resistant analogs of cAMP potentiates muscle contraction, and a phosphodiesterase inhibitor enhances the effect of MCC stimulation. Recordings from free-moving animals indicate that the MCC becomes activated by exposure of the animal to food stimuli, and that the activation parallels the presence of a food-arousal state. Food-arousal is characterized by enhanced strength and increased frequency of biting responses. Both these effects can result from activity of the MCC. Thus, in this system, modulatory synaptic actions function to provide the substrate for a type behavioral modulation.

Alteration of myoblast phenotype by dimethyl sulfoxide.

Application of dimethyl sulfoxide to proliferating L8 myoblasts (an established cell line of rat skeletal muscle) for 72 hr completely prevented fusion and induction of creatine phosphokinase (EC 2.7.3.2) activity (an indicator of muscle differentiation). The growth pattern changed from the usual sheets of randomly oriented cells to flattened, whorled monolayers of elongated fibroblast-like cells. By electron microscopy, rough endoplasmic reticulum increased and extracellular material appeared that had the morphologic and staining characteristics of collagen. After 120 hr in dimethyl sulfoxide-containing medium, the cells secreted about 6 times more collagen than untreated controls. Dimethyl sulfoxide was ineffective when applied to L8 cells just prior to fusion, and effects of dimethyl sulfoxide were not readily reversible unless treated cells were subcultured at low density.

Beta-adrenergic receptors and myogenesis.

The rat myogenic cell line, L8, contains a beta-adrenergic catecholamine-sensitive adenylate cyclase. Prior to cell fusion, and continuing thereafter, beta-adrenergic sites, as determined by the stereospecific binding of (125I)-hydroxybenqylpindolol, I1(125I)IHYP] increases from 470 to 2000 sites/cell. There is also an increase in adenylate cyclase (2-5 fold) and endogenous cAMP (5-30 fold) following stimulation by catecholamine. The dissociation constant (KD) of (125I)IHYP for unfused and fused cell-homogenates, as determined by estimation with Scatchard analysis, by direct determination at receptor concentrations well below the KD, or by association (4.6 X 10(8) M-1 min-1); and dissociation (0.028 min-1) kinetics; ranged from about 40 to 70 pM. The acquisition of beta-receptors prior to fusion in L8 cells may implicate this system in the regulation of myogenesis.