Adenosine deamination sustains dendritic cell activation in inflammation

Adenosine is a suppressive agent that protects the host from excessive tissue injury associated with strong inflammation. In tissue stress, higher levels of adenosine signal through adenosine receptors to exert strong anti-inflammatory effects, and thus protect host cells. Existing evidence also suggests that elevated adenosine potently down-regulates the activation of lymphocytes during inflammation. This notion, however, is in contrast with another basic observation that the immune system is highly activated precisely under the same circumstances against pathogens. In this study, we show that inflammatory responses of dendritic cells (DCs) are highly sensitive to adenosine suppression. However, they intrinsically carry high adenosine deaminase activity, which in turn degrades and removes adenosine from the surroundings, cutting off DCs from the suppression. This regulatory mechanism is important in DC responses to pathogen-associated molecular patterns and their activation of T cells. Our findings suggest a mechanism that DCs maintain their hyperreactive state in inflammation despite the general state of suppression, and reveal a regulatory role of adenosine deaminase in DC innate immune responses.     

Infection and persistence of rhesus monkey rhadinovirus in immortalized B-cell lines

Similar to its close relative human herpesvirus 8, rhesus monkey rhadinovirus (RRV) persists predominantly in B cells of its natural host. Rhesus monkey B-cell lines immortalized by the Epstein-Barr-related virus from rhesus monkeys (rhEBV) were used as targets for infection by RRV. These cultured B cells were susceptible to infection by RRV and continued to produce low titers of RRV for months of continuous culture. Infection by RRV did not detectably alter the growth rates of these B-cell lines when it was measured at standard or reduced serum concentrations. Depending on the cell line, 5 to 40% of the B cells stained positive for the RRV genome by fluorescence in situ hybridization (FISH). Most RRV-positive cells showed a fine punctate nuclear staining pattern consistent with latent infection, while a small minority of cells (0.2 to 1%) contained large, intensely staining nuclear foci consistent with productive, replicative infection. Greater than 90% of the cells were rhEBV genome positive in a pattern consistent with latent infection, and again only a small minority of cells showed a productive, replicative staining pattern. Dual, two-color FISH staining revealed coinfection of numerous cells with both RRV and rhEBV, but productive replication of RRV and rhEBV was always observed in separate cells, never in the same cell. Thus, productive replication of RRV is unlinked to that of rhEBV; factors that influence activation to productive replication act separately on RRV and rhEBV, even within the same cell. The percentage of B cells expressing green fluorescent protein (GFP) early after infection with a recombinant RRV containing a GFP reporter gene was dose dependent and at a low multiplicity of infection increased progressively over time until 14 to 17 days after infection. These results establish a naturalistic cell culture system for the study of infection and persistence by RRV in rhesus monkey B cells.     

The functional group approach to bioturbation: The effects of biodiffusers and gallery-diffusers of the Macoma balthica community on sediment oxygen uptake

The Macoma balthica community, which is widely distributed in intertidal soft sediments bordering the north Atlantic, is dominated by two functional groups with different sediment mixing modes: the biodiffusers M. balthica and Mya arenaria and the gallery-diffuser Nereis virens. To compare the effects of these two groups on sediment oxygen uptake rates, we used experimental microcosms with identical biovolumes to measure the influence of each species on oxygen uptake. The two biodiffusers had similar effects on oxygen uptake in spite of different space occupation and different feeding, ventilation and burrowing modes. Biodiffusers and gallery-diffusers had different effects on oxygen uptake. Periodic ventilation by the gallery-diffusers stimulated the oxygen uptake by the sediment more than the steady activities of the biodiffusers. Temporal variation in oxygen fluxes in bioturbated microcosms was linked to construction and maintenance of biogenic structures. The results confirm that the functional group approach to bioturbation is a useful tool for quantifying the effects of intertidal benthic communities on benthic fluxes.     

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3''-deoxyadenosine 5''-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.     

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.     

A Single Intravenous AAV9 Injection Mediates Bilateral Gene Transfer to the Adult Mouse Retina

Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.     

Islands within an island: Repeated adaptive divergence in a single population

Physical barriers to gene flow were once viewed as prerequisites for adaptive evolutionary divergence. However, a growing body of theoretical and empirical work suggests that divergence can proceed within a single population. Here we document genetic structure and spatially replicated patterns of phenotypic divergence within a bird species endemic to 250 km² Santa Cruz Island, California, USA. Island scrub‐jays (Aphelocoma insularis) in three separate stands of pine habitat had longer, shallower bills than jays in oak habitat, a pattern that mirrors adaptive differences between allopatric populations of the species’ mainland congener. Variation in both bill measurements was heritable, and island scrub‐jays mated nonrandomly with respect to bill morphology. The population was not panmictic; instead, we found a continuous pattern of isolation by distance across the east–west axis of the island, as well as a subtle genetic discontinuity across the boundary between the largest pine stand and adjacent oak habitat. The ecological factors that appear to have facilitated adaptive differentiation at such a fine scale—environmental heterogeneity and localized dispersal—are ubiquitous in nature. These findings support recent arguments that microgeographic patterns of adaptive divergence may be more common than currently appreciated, even in mobile taxonomic groups like birds.     

Relating macroinvertebrate community structure to environmental characteristics and sediment contamination at the scale of the St. Lawrence River

There is still no assessment of the impact of sediment chemicals and environmental conditions on macroinvertebrates at the scale of the St. Lawrence River. In order to assess these impacts in the fluvial section of the St. Lawrence River including the Montreal harbour, the community structure of macroinvertebrates using different taxonomic aggregations (genus and family) and taxa attributes (abundance, presence–absence, indicator taxa) was assessed. The goal of the study was to determine the indicator taxa of macroinvertebrates along the fluvial continuum and relate changes in macroinvertebrate community to sediment chemical conditions and environmental characteristics of habitats using variance partitioning. This study also evaluated which taxonomic level and taxa attributes of macroinvertebrates were the most suitable for bioassessment of quality of sediments and habitat environment in the St. Lawrence River. Four different macroinvertebrate assemblages were found distributed along the fluvial continuum using either abundance or presence–absence data and genus or family levels. Indicator taxa characteristic of the different macroinvertebrate communities were associated with the sediment contamination gradient. However, habitat environmental characteristics (water masses, sulphur and DOC in sediments) had more influence on macroinvertebrate assemblages than sediment contamination. Our study confirms that family level analysis can give information comparable to the genus level analysis using presence–absence or abundance of macroinvertebrates, yet a higher number of indicator taxa were detected at the genus level.