Cenicriviroc,a Novel CCR5 (R5) and CCR2 Antagonist,Shows In Vitro Activity against R5 Tropic HIV-2 Clinical Isolates

Background

Maraviroc activity against HIV-2, a virus naturally resistant to different HIV-1 antiretroviral drugs, has been recently demonstrated. The aim of this study was to assess HIV-2 susceptibility to cenicriviroc, a novel, once-daily, dual CCR5 and CCR2 antagonist that has completed Phase 2b development in HIV-1 infection.

Methods

Cenicriviroc phenotypic activity has been tested using a PBMC phenotypic susceptibility assay against four R5-, one X4- and one dual-tropic HIV-2 clinical primary isolates. All isolates were obtained by co-cultivation of PHA-activated PBMC from distinct HIV-2-infected CCR5-antagonist-naïve patients included in the French HIV-2 cohort and were previously tested for maraviroc susceptibility using the same protocol. HIV-2 tropism was determined by phenotypic assay using Ghost(3) cell lines.

Results

Regarding the 4 R5 HIV-2 clinical isolates tested, effective concentration 50% EC₅₀ for cenicriviroc were 0.03, 0.33, 0.45 and 0.98 nM, similar to those observed with maraviroc: 1.13, 0.58, 0.48 and 0.68 nM, respectively. Maximum percentages of inhibition (MPI) of cenicriviroc were 94, 94, 93 and 98%, similar to those observed with maraviroc (93, 90, 82, 100%, respectively). The dual- and X4-tropic HIV-2 strains were resistant to cenicriviroc with EC50 >1000 nM and MPI at 33% and 4%, respectively.

Conclusions

In this first study assessing HIV-2 susceptibility to cenicriviroc, we observed an in vitro activity against HIV-2 R5-tropic strains similar to that observed with maraviroc. Thus, cenicriviroc may offer a once-daily treatment opportunity in the limited therapeutic arsenal for HIV-2. Clinical studies are warranted.     

Association of SNAREs and Calcium Channels with the Borders of Cytoskeletal Cages Organizes the Secretory Machinery in Chromaffin Cells

In chromaffin cells, SNARE proteins, forming the basic exocytotic machinery are present in membrane clusters of 500–600 nm in diameter. These microdomains containing both SNAP-25 and syntaxin-1 are dynamic and the expression of altered forms of SNAREs modifies not only their motion but also the mobility of the associated granules. It is also clear that SNARE microdomain location defines the place for individual vesicle fusion and that the alteration of cluster dynamics affects the fusion process itself. Interestingly, these SNARE patches colocalize with the borders of F-actin cages forming the cytoskeletal cortical network, and these borders also contain clusters of L- and P/Q type calcium channels. The organization of the secretory machinery in association with the borders of cytoskeletal cages seems to be an effective way to promote fast coupling between calcium entry and catecholamine release as demonstrated with the use of mathematical secretory models.     

Regulation of GSK3 isoforms by phosphatases PP1 and PP2A

Dephosphorylation of phospho GSK3 isoforms, from COS-7 cells, was determined in vitro and in cultured cells in the absence or the presence of okadaic acid and lithium. Our results indicate a preferential dephosphorylation of phospho GSK3α by PP2A phosphatase, whereas dephosphorylation of phospho GSK3β mainly takes place by PP1 phosphatase.     

Dynamic loading stimulates chondrocyte biosynthesis when encapsulated in charged hydrogels prepared from poly(ethylene glycol) and chondroitin sulfate

This study aimed to elucidate the role of charge in mediating chondrocyte response to loading by employing synthetic 3D hydrogels. Specifically, neutral poly(ethylene glycol) (PEG) hydrogels were employed where negatively charged chondroitin sulfate (ChS), one of the main extracellular matrix components of cartilage, was systematically incorporated into the PEG network at 0%, 20% or 40% to control the fixed charge density. PEG hydrogels were employed as a control environment for extracellular events which occur as a result of loading, but which are not associated with a charged matrix (e.g., cell deformation and fluid flow). Freshly isolated bovine articular chondrocytes were embedded in the hydrogels and subject to dynamic mechanical stimulation (0.3 Hz, 15% amplitude strains, 6 h) and assayed for nitric oxide production, cell proliferation, proteoglycan synthesis, and collagen deposition. In the absence of loading, incorporation of charge inhibited cell proliferation by ~ 75%, proteoglycan synthesis by ~ 22–50% depending on ChS content, but had no affect on collagen deposition. Dynamic loading had no effect on cellular responses in PEG hydrogels. However, dynamically loading 20% ChS gels inhibited nitrite production by 50%, cell proliferation by 40%, but stimulated proteoglycan and collagen deposition by 162% and 565%, respectively. Dynamic loading of 40% ChS hydrogels stimulated nitrite production by 62% and proteoglycan synthesis by 123%, but inhibited cell proliferation by 54% and collagen deposition by 52%. Upon removing the load and culturing under free-swelling conditions for 36 h, the enhanced matrix synthesis observed in the 20% ChS gels was not maintained suggesting that loading is necessary to stimulate matrix production. In conclusion, extracellular events associated with a charged matrix have a dramatic affect on how chondrocytes respond to mechanical stimulation within these artificial 3D matrices suggesting that streaming potentials and/or dynamic changes in osmolarity may be important regulators of chondrocytes while cell deformation and fluid flow appear to have less of an effect.     

Abiotic influences on embryo growth: statoliths as experimental tools in the squid early life history

Statolith size and growth was used to determine the influence of abiotic factors on the growth of Loligo vulgaris and Sepioteuthis australis embryos. Recently spawned egg masses collected from the field were incubated in the laboratory under different levels of light intensity, photoperiod, or short periods of low salinity (30‰). Double tetracycline staining was used to follow statolith growth. In L. vulgaris constant light conditions produced significantly slower growth in the embryonic statoliths and embryos held at summer photoperiod had slower statolith growth than those held at winter photoperiods. However once they hatched out there was no evidence that photoperiod affected statolith growth. After hatching, in all photoperiods statolith growth rates decreased in comparison with late embryonic rates. In S. australis embryos, differences between the high and medium light intensities for summer and intermediate photoperiods were found, suggesting that under summer incubation temperature, longer daylengths at medium light intensity favoured higher statolith growth for this species. In comparison to controls, slower statolith growth in S. australis embryos due to low salinity only occurred when exposed for 72 h. Comparison with previous studies indicates that temperature seems to be the main abiotic factor influencing statolith growth during early stages, however, interactions among all abiotic factors needs to be determined as well as the unknown influence of other isolated factors, e.g., oxygen concentration within the egg mass.     

Ethical and welfare considerations when using cephalopods as experimental animals

When using cephalopods as experimental animals, a number of factors, including morality, quality of information derived from experiments, and public perception, drives the motivation to consider welfare issues. Refinement of methods and techniques is a major step in ensuring protection of cephalopod welfare in both laboratory and field studies. To this end, existing literature that provides details of methods used in the collection, handling, maintenance, and culture of a range of cephalopods is a useful starting point when refining and justifying decisions about animal welfare. This review collates recent literature in which authors have used cephalopods as experimental animals, revealing the extent of use and diversity of cephalopod species and techniques. It also highlights several major issues when considering cephalopod welfare; how little is known about disease in cephalopods and its relationship to senescence and also how to define objective endpoints when animals are stressed or dying as a result of the experiment.