Evidence of Rise in Rabies Cases in Southern Malawi - Better Preventative Measures Are Urgently Required

We describe five children who died of clinical rabies in a three month period (September to November 2011) in the Queen Elizabeth Central Hospital. From previous experience and hospital records, this number of cases is higher than expected. We are concerned that difficulty in accessing post-exposure prophylaxis (PEP) rabies vaccine may be partly responsible for this rise. We advocate:(a) prompt course of active immunisation for all patients with significant exposure to proven or suspected rabid animals. (b) the use of an intradermal immunisation regime that requires a smaller quantity of the vaccine than the intramuscular regime and gives a better antibody response. (c) improved dog rabies control measures     

Quantitative analysis of neuropeptide Y receptor association with ��-arrestin2 measured by bimolecular fluorescence complementation

Background and purpose:

β-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and β-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein–protein interactions.

Experimental approach:

Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and β-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor–β-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/β-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis.

Key results:

NPY stimulation promoted association between Y1–Yc and β-arrestin2–Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor–YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, β-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for β-arrestin2. Receptor mutagenesis combined with β-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support β-arrestin2 association, and that Y2 receptor–β-arrestin2 interaction was enhanced by the intracellular loop mutation H155P.

Conclusions and implications:

The BiFC approach quantifies Y receptor ligand pharmacology focused on the β-arrestin2 pathway, and provides insight into mechanisms of β-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein–protein interaction.     

The melanocortin MC1 receptor agonist BMS-470539 inhibits leucocyte trafficking in the inflamed vasculature

Background and purpose:

Over three decades of research evaluating the biology of melanocortin (MC) hormones and synthetic peptides, activation of the MC type 1 (MC₁) receptor has been identified as a viable target for the development of novel anti-inflammatory therapeutic agents. Here, we have tested a recently described selective agonist of MC₁ receptors, BMS-470539, on leucocyte/post-capillary venule interactions in murine microvascular beds.

Experimental approach:

Intravital microscopy of two murine microcirculations were utilized, applying two distinct modes of promoting inflammation. The specificity of the effects of BMS-470539 was assessed using mice bearing mutant inactive MC₁ receptors (the recessive yellow e/e colony).

Key results:

BMS-470539, given before an ischaemia–reperfusion protocol, inhibited cell adhesion and emigration with no effect on cell rolling, as assessed 90 min into the reperfusion phase. These properties were paralleled by inhibition of tissue expression of both CXCL1 and CCL2. Confocal investigations of inflamed post-capillary venules revealed immunostaining for MC₁ receptors on adherent and emigrated leucocytes. Congruently, the anti-inflammatory properties of BMS-470539 were lost in mesenteries of mice bearing the inactive mutant MC₁ receptors. Therapeutic administration of BMS-470539 stopped cell emigration, but did not affect cell adhesion in the cremasteric microcirculation inflamed by superfusion with platelet-activating factor.

Conclusions and implications:

Activation of MC₁ receptors inhibited leucocyte adhesion and emigration. Development of new chemical entities directed at MC₁ receptors could be a viable approach in the development of novel anti-inflammatory therapeutic agents with potential application to post-ischaemic conditions.