Identification of the ancestral killer immunoglobulin-like receptor gene in primates

BACKGROUND: Killer Immunoglobulin-like Receptors (KIR) are essential immuno-surveillance molecules. They are expressed on natural killer and T cells, and interact with human leukocyte antigens. KIR genes are highly polymorphic and contribute vital variability to our immune system. Numerous KIR genes, belonging to five distinct lineages, have been identified in all primates examined thus far and shown to be rapidly evolving. Since few KIR remain orthologous between species, with only one of them, KIR2DL4, shown to be common to human, apes and monkeys, the evolution of the KIR gene family in primates remains unclear. RESULTS: Using comparative analyses, we have identified the ancestral KIR lineage (provisionally named KIR3DL0) in primates. We show KIR3DL0 to be highly conserved with the identification of orthologues in human (Homo sapiens), common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), rhesus monkey (Macaca mulatta) and common marmoset (Callithrix jacchus). We predict KIR3DL0 to encode a functional molecule in all primates by demonstrating expression in human, chimpanzee and rhesus monkey. Using the rhesus monkey as a model, we further show the expression profile to be typical of KIR by quantitative measurement of KIR3DL0 from an enriched population of natural killer cells. CONCLUSION: One reason why KIR3DL0 may have escaped discovery for so long is that, in human, it maps in between two related leukocyte immunoglobulin-like receptor clusters outside the known KIR gene cluster on Chromosome 19. Based on genomic, cDNA, expression and phylogenetic data, we report a novel lineage of immunoglobulin receptors belonging to the KIR family, which is highly conserved throughout 50 million years of primate evolution

Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution

<p>Abstract</p> <p>Background</p> <p>Vaccination of macaques with live attenuated simian immunodeficiency virus (SIV) provides significant protection against the wild-type virus. The use of a live attenuated human immunodeficiency virus (HIV) as AIDS vaccine in humans is however considered unsafe because of the risk that the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. We earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (dox). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient dox administration. Since the effectiveness and safety of such a conditionally live virus vaccine should be tested in macaques, we constructed a similar dox-dependent SIV variant. The Tat-TAR transcription control mechanism in this virus was inactivated through mutation and functionally replaced by the dox-inducible Tet-On regulatory system. This SIV-rtTA variant replicated in a dox-dependent manner in T cell lines, but not as efficiently as the parental SIVmac239 strain. Since macaque studies will likely require an efficiently replicating variant, we set out to optimize SIV-rtTA through in vitro viral evolution.</p> <p>Results</p> <p>Upon long-term culturing of SIV-rtTA, additional nucleotide substitutions were observed in TAR that affect the structure of this RNA element but that do not restore Tat binding. We demonstrate that the bulge and loop mutations that we had introduced in the TAR element of SIV-rtTA to inactivate the Tat-TAR mechanism, shifted the equilibrium between two alternative conformations of TAR. The additional TAR mutations observed in the evolved variants partially or completely restored this equilibrium, which suggests that the balance between the two TAR conformations is important for efficient viral replication. Moreover, SIV-rtTA acquired mutations in the U3 promoter region. We demonstrate that these TAR and U3 changes improve viral replication in T-cell lines and macaque peripheral blood mononuclear cells (PBMC) but do not affect dox-control.</p> <p>Conclusion</p> <p>The dox-dependent SIV-rtTA variant was optimized by viral evolution, yielding variants that can be used to test the conditionally live virus vaccine approach and as a tool in SIV biology studies and vaccine research.</p

Hydration of dicalcium silicate and diffusion through neo-formed calcium-silicate-hydrates at weathered surfaces control the long-term leaching behaviour of basic oxygen furnace (BOF) steelmaking slag

Alkalinity generation and toxic trace metal (such as vanadium) leaching from basic oxygen furnace (BOF) steel slag particles must be properly understood and managed by pre-conditioning if beneficial reuse of slag is to be maximised. Water leaching under aerated conditions was investigated using fresh BOF slag at three different particle sizes (0.5–1.0, 2–5 and 10 × 10 × 20 mm blocks) and a 6-month pre-weathered block. There were several distinct leaching stages observed over time associated with different phases controlling the solution chemistry: (1) free-lime (CaO) dissolution (days 0–2); (2) dicalcium silicate (Ca₂SiO₄) dissolution (days 2–14) and (3) Ca–Si–H and CaCO₃ formation and subsequent dissolution (days 14–73). Experiments with the smallest size fraction resulted in the highest Ca, Si and V concentrations, highlighting the role of surface area in controlling initial leaching. After ~2 weeks, the solution Ca/Si ratio (0.7–0.9) evolved to equal those found within a Ca–Si–H phase that replaced dicalcium silicate and free-lime phases in a 30- to 150-μm altered surface region. V release was a two-stage process; initially, V was released by dicalcium silicate dissolution, but V also isomorphically substituted for Si into the neo-formed Ca–Si–H in the alteration zone. Therefore, on longer timescales, the release of V to solution was primarily controlled by considerably slower Ca–Si–H dissolution rates, which decreased the rate of V release by an order of magnitude. Overall, the results indicate that the BOF slag leaching mechanism evolves from a situation initially dominated by rapid hydration and dissolution of primary dicalcium silicate/free-lime phases, to a slow diffusion limited process controlled by the solubility of secondary Ca–Si–H and CaCO₃ phases that replace and cover more reactive primary slag phases at particle surfaces

Alkaline residues and the environment: A review of impacts, management practices and opportunities

Around two billion tonnes of alkaline residues are produced globally each year by industries such as steel production, alumina refining and coal-fired power generation, with a total production estimate of 90 billion tonnes since industrialization. These wastes are frequently stored in waste piles or landfills, and can be an environmental hazard if allowed to generate dust, or if rainwater infiltrates the waste. This review will focus on the environmental impacts associated with alkaline residues, with emphasis on the leachates produced by rainwater ingress. Many alkaline industrial wastes can produce leachates that are enriched with trace metals that form oxyanions (e.g. As, Cr, Mo, Se, V), which can be very mobile in alkaline water. The management options for the residues and their leachates are also discussed, distinguishing active and passive treatment options. Potential reuses of these materials, in construction materials, as agricultural amendments, and in environmental applications are identified. The mechanisms of carbon sequestration by alkaline residues are assessed, and the potential for enhancing its rate as a climate change off-setting measure for the industry is evaluated. The potential for recovery of metals critical to e-technologies, such as vanadium, cobalt, lithium and rare earths, from alkaline residues is considered. Finally research needs are identified, including the need to better understand the biogeochemistry of highly alkaline systems in order to develop predictable passive remediation and metal recovery technologies

Induction of a virus-specific effector–memory CD4+ T cell response by attenuated SIV infection

We investigated simian immunodeficiency virus (SIV)-specific CD4(+) T cell responses in rhesus macaques chronically infected with attenuated or pathogenic SIV strains. Analysis of SIVΔnef-infected animals revealed a relatively high frequency of SIV-specific CD4(+) T cells representing 4–10% of all CD4(+) T lymphocytes directed against multiple SIV proteins. Gag-specific CD4(+) T cells in wild-type SIV-infected animals were 5–10-fold lower in frequency and inversely correlated with the level of plasma viremia. SIV-specific CD4(+) cells from SIVΔnef animals were predominantly CD27(−)CD28(−)CD45RA(low)CCR7(−)CCR5(−), consistent with an effector–memory subset, and included a fully differentiated CD45RA(+)CCR7(−) subpopulation. In contrast, SIV-specific CD4(+) T cells from SIV-infected animals were mostly CD27(+)CD28(+)CD45RA(−)CCR7(+)CCR5(+), consistent with an early central memory phenotype. The CD45RA(+)CCR7(−)CD4(+) subset from SIVΔnef animals was highly enriched for effector CD4(+) T cells, as indicated by the perforin expression and up-regulation of the lysosomal membrane protein CD107a after SIV Gag stimulation. SIV-specific CD4(+) T cells in attenuated SIV-infected animals were increased in frequency in bronchioalveolar lavage and decreased in lymph nodes, consistent with an effector–memory T cell population. The ability of SIVΔnef to induce a high frequency virus-specific CD4(+) T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains

High Specific Infectivity of Plasma Virus from the Pre-Ramp-Up and Ramp-Up Stages of Acute Simian Immunodeficiency Virus Infection▿

To define the ratio of simian immunodeficiency virus (SIV) RNA molecules to infectious virions in plasma, a ramp-up-stage plasma pool was made from the earliest viral RNA (vRNA)-positive plasma samples (collected approximately 7 days after inoculation) from seven macaques, and a set-point-stage plasma pool was made from plasma samples collected 10 to 16 weeks after peak viremia from seven macaques; vRNA levels in these plasma pools were determined, and serial 10-fold dilutions containing 1 to 1,500 vRNA copies/ml were made. Intravenous (i.v.) inoculation of a 1-ml aliquot of diluted ramp-up-stage plasma containing 20 vRNA copies infected 2 of 2 rhesus macaques, while for the set-point-stage plasma, i.v. inoculation with 1,500 vRNA copies was needed to transmit infection. Further, when the heat-inactivated set-point-stage plasma pool was mixed with ramp-up-stage virions, infection of inoculated macaques was blocked. Notably, 2 of 2 animals inoculated with 85 ml of a pre-ramp-up plasma pool containing <3 SIV RNA copies/ml developed SIV infections characterized by high levels of viral replication, demonstrating that “vRNA-negative” plasma collected from macaques in the pre-ramp-up stage is infectious. Furthermore, there is a high ratio of infectious virions to total virions in ramp-up-stage plasma (between 1:1 and 1:10) and a lower ratio in set-point-stage plasma (between 1:75 and 1:750). Heat-inactivated chronic-stage plasma can “neutralize” the highly infectious ramp-up-stage virions. These findings have implications for the understanding of the natural history of SIV and human immunodeficiency virus infection and transmission

Retroviral Recombination In Vivo: Viral Replication Patterns and Genetic Structure of Simian Immunodeficiency Virus (SIV) Populations in Rhesus Macaques after Simultaneous or Sequential Intravaginal Inoculation with SIVmac239Δvpx/Δvpr and SIVmac239Δnef

To characterize the occurrence, frequency, and kinetics of retroviral recombination in vivo, we intravaginally inoculated rhesus macaques, either simultaneously or sequentially, with attenuated simian immunodeficiency virus (SIV) strains having complementary deletions in their accessory genes and various degrees of replication impairment. In monkeys inoculated simultaneously with SIVmac239Δvpx/Δvpr and SIVmac239Δnef, recombinant wild-type (wt) virus and wild-type levels of plasma viral RNA (vRNA) were detected in blood by 2 weeks postinoculation. In monkeys inoculated first with SIVmac239Δvpx/Δvpr and then with SIVmac239Δnef, recombination occurred but was associated with lower plasma vRNA levels than plasma vRNA levels seen for monkeys inoculated intravaginally with wt SIVmac239. In one monkey, recombination occurred 6 weeks after the challenge with SIVmac239Δnef when plasma SIVmac239Δvpx/Δvpr RNA levels were undetectable. In monkeys inoculated first with the more highly replicating strain, SIVmac239Δnef, and then with SIVmac239Δvpx/Δvpr, wild-type recombinant virus was not detected in blood or tissues. Instead, a virus that had repaired the deletion in the nef gene by a compensatory mutation was found in one animal. Overall, recombinant SIV was eventually found in four of six animals intravaginally inoculated with the two SIVmac239 deletion mutants. These findings show that recombination can occur readily in vivo after mucosal SIV exposure and thus contributes to the generation of viral genetic diversity and enhancement of viral fitness

Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution-1

From 13 independent cultures at different times and the LTR region was subsequently PCR amplified and sequenced. The number of the culture (#) and the day of sampling are indicated on the left. The -90 to +130 U3/R region is shown with +1 indicating the transcription initiation site. The Sp1 and TATA box are shown in grey. The mutations that were introduced in TAR to abolish Tat-responsiveness are underlined. The additional nucleotide substitutions and deletions (Δ) observed in the SIV-rtTA cultures are indicated.<p><b>Copyright information:</b></p><p>Taken from "Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution"</p><p>Retrovirology 2008;5():44-44.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2443169.</p><p></p

Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution-3

Constructs corresponding to the original and evolved SIV-rtTA variants and an rtTA-expressing plasmid. After two days of culturing with 0 to 1000 ng/ml dox, the intracellular luciferase level, which reflects promoter activity, was measured. The error bar represents the standard deviation (SD) for 3 to 8 experiments (B) To assay Tat responsiveness, C33A cells were transfected with the promoter/luciferase plasmids and 0 to 50 ng SIV Tat-expressing plasmid. Two days after transfection, the promoter activity was analyzed by measuring the intracellular luciferase activity. The error bar represents the SD for 2 to 4 experiments. (C) 293T cells were transfected with the SIV-rtTA proviral constructs and cultured for two days with or without dox. Virus production was quantified by measuring the CA-p27 level in the culture supernatant. The error bar represents the standard deviation for 2 experiments.<p><b>Copyright information:</b></p><p>Taken from "Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution"</p><p>Retrovirology 2008;5():44-44.</p><p>Published online 5 Jun 2008</p><p>PMCID:PMC2443169.</p><p></p