Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry

CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for inhibiting HIV infection. We exploited the characteristics of the chemokine analog PSC-RANTES (N-α-(n-nonanoyl)-des-Ser(1)-[l-thioprolyl(2), l-cyclohexylglycyl(3)]-RANTES(4-68)), which displays potent anti-HIV-1 activity. We show that native chemokines fail to prevent high-affinity binding of PSC-RANTES, analog-mediated calcium release (in desensitization assays), and analog-mediated CCR5 internalization. These results indicate that a pool of spare CCR5 may bind PSC-RANTES but not native chemokines. Improved recognition of CCR5 by PSC-RANTES may explain why the analog promotes higher amounts of β-arrestin 2·CCR5 complexes, thereby increasing CCR5 down-regulation and HIV-1 inhibition. Together, these results highlight that spare CCR5, which might permit HIV-1 to escape from chemokines, should be targeted for efficient viral blockade.     

Deep-time phylogenetic clustering of extinctions in an evolutionarily dynamic clade (Early Jurassic ammonites)

Conservation biologists and palaeontologists are increasingly investigating the phylogenetic distribution of extinctions and its evolutionary consequences. However, the dearth of palaeontological studies on that subject and the lack of methodological consensus hamper our understanding of that major evolutionary phenomenon. Here we address this issue by (i) reviewing the approaches used to quantify the phylogenetic selectivity of extinctions and extinction risks; (ii) investigating with a high-resolution dataset whether extinctions and survivals were phylogenetically clustered among early Pliensbachian (Early Jurassic) ammonites; (iii) exploring the phylogenetic and temporal maintenance of this signal. We found that ammonite extinctions were significantly clumped phylogenetically, a pattern that prevailed throughout the 6.6 Myr-long early Pliensbachian interval. Such a phylogenetic conservatism did not alter--or may even have promoted--the evolutionary success of this major cephalopod clade. However, the comparison of phylogenetic autocorrelation among studies remains problematic because the notion of phylogenetic conservatism is scale-dependent and the intensity of the signal is sensitive to temporal resolution. We recommend a combined use of Moran's I, Pearson's ? and Fritz and Purvis' D statistics because they highlight different facets of the phylogenetic pattern of extinctions and/or survivals.     

Cardiac shock wave therapy: assessment of safety and new insights into mechanisms of tissue regeneration

Although low-energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non-invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four-month-old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin-I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c-kit-positive, Ki67-positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65-fold more numerous in the treated myocardium. In conclusion, non-invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.     

Potential role of phospholipase D2 in increasing interleukin-2 production by T-lymphocytes through activation of mitogen-activated protein kinases ERK1/ERK2

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of phosphatidic acid (PA), which is itself a source of diacylglycerol (DAG). These two versatile lipid second messengers are at the centre of a phospholipid signalling network and as such are involved in several cellular functions. However, their role in T-cell activation and functions are still enigmatic. In order to elucidate this role, we generated a human and a murine T-cell line that stably overexpressed the PLD2 isoform. Analysis of the Ras-MAPK pathway upon phorbol myristate acetate (PMA) and ionomycin stimulation revealed that PLD2 promoted an early and sustained increase in ERK1/2 phosphorylation in both cell lines. This response was inhibited by 1-butanol, a well known distracter of PLD activity, or upon overexpression of a dominant negative PLD2, and it was concomitant with a boost of PA/DAG production. As a functional consequence of this PLD2-dependent MAPK activation, interleukin-2 production evoked by PMA/ionomycin stimulation or CD3/CD28 engagement was enhanced in the two T-cell lines overexpressing PLD2. Thus, PLD2 emerged as an early player upstream of the Ras-MAPK-IL-2 pathway in T-cells via PA and DAG production, raising new possibilities of pharmacological manipulation in immune disorders.     

Naturally occurring melanomas in dogs as models for non‐UV pathways of human melanomas

Spontaneously occurring melanomas are frequent in dogs. They appear at the same localizations as in humans, i.e. skin, mucosal sites, nail matrix and eyes. They display variable behaviors: tumors at oral localizations are more frequent and aggressive than at other anatomical sites. Interestingly, dog melanomas are associated with strong breed predispositions and overrepresentation of black‐coated dogs. Epidemiological analysis of 2350 affected dogs showed that poodles are at high risk of developing oral melanoma, while schnauzers or Beauce shepherds mostly developped cutaneous melanoma. Clinical and histopathological analyses were performed on a cohort of 153 cases with a 4‐yr follow‐up. Histopathological characterization showed that most canine tumors are intradermal and homologous to human rare morphological melanomas types – ‘nevocytoid type’ and ‘animal type’‐. Tumor cDNA sequencing data, obtained from 95 dogs for six genes, relevant to human melanoma classification, detected somatic mutations in oral melanoma, in NRAS and PTEN genes, at human hotspot sites, but not in BRAF. Altogether, these findings support the relevance of the dog model for comparative oncology of melanomas, especially for the elucidation of non‐UV induced pathways.     

Sperm motility and fertilizing ability of frozen spermatozoa of males (XY) and neomales (XX) of perch (Perca fluviatilis)

The objective of this study was to freeze sperm of sex‐reversed females (neomales) of perch and to test their fertilization ability. Sperm used was testicular (TSN), collected from females that have been inverted by means of externally administered 17‐alpha methyltestosterone. Sperm collected from intact males (SSNM) of the same origin were used as control. Prior to freezing, both TSN and SSNM were diluted into 300 mm glucose solution at the ratio of 1 : 6 and DMSO was used as cryoprotectant (10% final concentration). Crypreservation was performed in 0.5 ml straws placed into a polystyrene box, three cm above the liquid nitrogen level for 10 min and thereafter transferred fully into liquid nitrogen. Samples were thawed in 40°C water bath for 8 s and used for the fertilization experiments. Spermatozoa concentration of fresh TSN and SSNM were estimated with 45.3 × 10⁹ and 37.8 × 10⁹ spermatozoa ml^?1, respectively. Both sperm velocity and motility showed significant decreases in the TSN (134.6 μm s^?1 and 12.8%) compared to the SSNM (203.2 μm s^?1 and 94.7%) at 10 s after sperm activation. However, no differences were observed in terms of hatching rates between fresh TSN and SSNM (42.5 vs 49.3%) at fertilization densities of 12 × 10⁵ spermatozoa per egg. Frozen/thawed SSNM exhibited similar hatching rates at 12 × 10⁵ and 2.4 × 10⁵ spermatozoa per egg (37.2% vs 29.1%). Hatching rates for frozen/thawed TSN were about 7.3% with 12 × 10⁵ spermatozoa per egg and did not show any difference at 2.4 × 10⁵ spermatozoa per egg (6.6%). Stripped sperm of normal perch can be successfully frozen. Squeezing of the testes is not a good method for collection of testicular sperm resulting into low velocity, motility and hatching rate. To understand the influences of neomales on sperm quality on reproductive success further studies should be performed addressing a full assay of motility and fertility criteria when using stripped sperm from normal males and neomales. Additionally, the results indicate that many of sex reversed perch neomales are not able to release sperm and that for further studies some well spermiating neomales must to be selected.     

Female house sparrows "count on" male genes: experimental evidence for MHC-dependent mate preference in birds

Background  

Females can potentially assess the quality of potential mates using their secondary sexual traits, and obtain "good genes" that increase offspring fitness. Another potential indirect benefit from mating preferences is genetic compatibility, which does not require extravagant or viability indicator traits. Several studies with mammals and fish indicate that the genes of the major histocompatibility complex (MHC) influence olfactory cues and mating preferences, and such preferences confer genetic benefits to offspring. We investigated whether individual MHC diversity (class I) influences mating preferences in house sparrows (Passer domesticus).     

General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines

Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcgamma receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 x 10(4) units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future.