Workshop on Use of Intravenous Immunoglobulin in Hand,Foot and Mouth Disease in Southeast Asia

The South East Asia Infectious Disease Clinical Research Network convened subject matter experts at a workshop to make consensus recommendations for study design of a clinical trial for use of intravenous immunoglobulin (IVIg) in severe hand, foot and mouth disease (HFMD). HFMD is a highly contagious emerging infection among children in the region, a small proportion of whom develop neurologic and cardiopulmonary complications with high case-fatality rates. The use of IVIg for treatment of severe disease is widespread and a part of local, national, and international guidelines, but no clinical evidence warrants the use of this drug, which is expensive and has potentially serious side effects. During a 2-day workshop in March 2014, a group of HFMD experts reviewed the current evidence related to use of IVIg in HFMD and discussed potential study design, feasibility, inclusion and exclusion criteria, sample size, primary and secondary endpoints, and subsidiary studies for a randomized, placebo-controlled trial.     

Virology, epidemiology, pathogenesis, and control of enterovirus 71

First isolated in California, USA, in 1969, enterovirus 71 (EV71) is a major public health issue across the Asia-Pacific region and beyond. The virus, which is closely related to polioviruses, mostly affects children and causes hand, foot, and mouth disease with neurological and systemic complications. Specific receptors for this virus are found on white blood cells, cells in the respiratory and gastrointestinal tract, and dendritic cells. Being an RNA virus, EV71 lacks a proofreading mechanism and is evolving rapidly, with new outbreaks occurring across Asia in regular cycles, and virus gene subgroups seem to differ in clinical epidemiological properties. The pathogenesis of the severe cardiopulmonary manifestations and the relative contributions of neurogenic pulmonary oedema, cardiac dysfunction, increased vascular permeability, and cytokine storm are controversial. Public health interventions to control outbreaks involve social distancing measures, but their effectiveness has not been fully assessed. Vaccines being developed include inactivated whole-virus, live attenuated, subviral particle, and DNA vaccines.     

Sex differences in neuronal morphology in the killifish hypothalamus

This study examined the neuroarchitecture of the male and female killifish (Fundulus heteroclitus) hypothalamus to evaluate whether sexual dimorphism of this brain region exists in fishes as it does in mammals and other vertebrates. The rostral medulla, a brain region distinct from the hypothalamic-pituitary-gonadal axis, was also examined to determine if any observed differences were region-specific. With the use of Golgi-Cox impregnation, five dendritic characteristics were measured from neurons of both the hypothalamus and medulla including: spine density, number of branch points, dendrite length, surface area and volume. Dendritic spines are associated with excitatory synapses, and changes in density are associated with a variety of normal and pathological changes. Consistent with mammalian studies, we found that adult female killifish have 25% greater dendritic spine densities in the hypothalamus than male killifish (densities of 0.34+/-0.06 microm-1 and 0.25+/-0.08 microm-1, respectively). By contrast, no statistically significant difference between males and females was detected in spine densities in the rostral medulla. This finding supports the conclusion that hypothalamic sexual dimorphism is conserved in killifish.     

Canonical and noncanonical Wnts use a common mechanism to activate completely unrelated coreceptors

Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.     

Relationship between the intracellular effects of camptothecin and the inhibition of DNA topoisomerase I in cultured L1210 cells

Results of filter elution assays of lesions produced in the DNA of cultured L1210 cells by the antineoplastic alkaloid camptothecin support the notion that topoisomerase I is an intracellular target of this drug. One to 10 microM camptothecin induced DNA single-strand, but not double-strand, breaks when incubated with intact cells or with their isolated nuclei. Approximately one half of the strand breakage was protein concealed, as judged by filter elution. Camptothecin-induced, protein-concealed DNA strand breaks disappeared rapidly after drug removal. DNA-protein cross-links were generated by camptothecin with frequencies approximately equal to those of protein-concealed DNA strand breaks. It is likely that camptothecin can inhibit topoisomerase I in intact cells in a manner similar to that in which other antineoplastic agents such as amsacrine or teniposide inhibit topoisomerase II. DNA-breaking lesions other than those resulting from trapped topoisomerase I-DNA complexes may also be generated by camptothecin. The yields of DNA strand breaks induced by camptothecin, amsacrine, or teniposide were approximately doubled when cells were incubated for 16 h with 3-aminobenzamide, an inhibitor of poly(ADP ribosylation) of proteins, prior to 1-h exposure to the antineoplastic compounds. 3-Aminobenzamide also enhanced the cytotoxic action of camptothecin, amsacrine, and teniposide. These results suggest that protein-concealed strand breaks can be lethal lesions and that intracellular topoisomerase I and II activity may be regulated coordinately through poly(ADP ribosylation).     

Post soluble binding to the leukotriene D4 receptor from guinea pig lung membranes

Guinea pig lung membranes were extracted with 1% digitonin and yielded a preparation that contained soluble leukotriene D4 (LTD4) receptor. Specific binding of the high affinity radiolabeled receptor antagonist [3H]ICI-198615 to the soluble LTD4 receptor was time dependent and reversible. The dissociation constant (Kd) and the density (Bmax) of [3H]ICI-198615 binding to the soluble LTD4 receptor was 0.2 +/- 0.08 nM and 380 +/- 40 fmol/mg of protein, respectively. Radioligand competition studies showed several classes of structurally diverse, functionally defined, receptor antagonists competed with [3H]ICI-198615 binding to the soluble receptor. The rank order of potency and specificity of these antagonists in binding to the soluble receptor were equivalent to those determined from the membrane-bound receptor binding assay and from the smooth muscle contraction assay. Binding of LTD4 to the soluble receptor was observed, in the competition assay, only in the low affinity state (Ki = 2 microM). Size-exclusion chromatography of the soluble LTD4 receptor showed that the apparent molecular weight of the LTD4 receptor in digitonin micelle was approximately 300,000.     

Characterization of [3H]leukotriene D4 binding sites in guinea-pig ventricular myocardium

[3H]Leukotriene (LT) D4 was used to identify specific LTD4 binding sites in guinea-pig ventricular myocardial membranes. High-performance liquid chromatography analyses indicated that, in the presence of the gamma-glutamyl transpeptidase inhibitor L-serine-borate (80 mM), less than 3% of membrane-bound [3H]LTD4 was converted to [3H]LTC4 or [3H]LTE4 at 30 degrees C. The specific [3H] LTD4 binding, assayed in the presence of 80 mM L-serine-borate, reached a stable steady state within 45 min at 30 degrees C (pH 7.5). A monophasic Scatchard plot of saturation binding data yielded an apparent dissociation constant (Kd) of 3.4 +/- 2.1 nM and a maximum number of binding sites of 850 +/- 91 fmol/mg of protein. Competition binding studies with [3H]LTD4, synthetic 5S, 6R-LTD4 (LTD4) and its diastereoisomer 5R,6S-LTD4, LTE4, LTC4 and the putative LT antagonists FPL 55712, 4R-hydroxy-5S-1-cysteinylglycine-6Z-nonadecanoic acid (2-nor-LTD1) and SKF 88046 demonstrated a potency order of LTD4 greater than LTE4 greater than LTC4 greater than 5R,6S-LTD4 much greater than 2-nor-LTD1. FPL 55712 and SKF 88046 were ineffective in displacing the specific [3H]LTD4 binding. Pretreatment of the heart membranes with the sulfhydryl reducing reagent dithiothreitol decreased the specific [3H]LTD4 binding in a concentration-dependent manner. Scatchard analyses of saturation isotherms indicated that 0.3 mM dithiothreitol pretreatment of heart membranes decreased the maximum number of binding sites of the [3H]LTD4 binding to 368 +/- 61 fmol/mg of protein with minimal effects on the apparent Kd.(ABSTRACT TRUNCATED AT 250 WORDS)