Activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (PIV5) P/V mutant

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of na?ve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity.     

The distribution–abundance (density) relationship: its form and causes in a tropical mammal order, Primates

Aim Across a wide variety of organisms, taxa with high local densities (abundance) have large geographical ranges (distributions). We use primatology's detailed knowledge of its taxon to investigate the form and causes of the relationship in, unusually for macroecological analysis, a tropical taxon. Location Africa, Central and South America, Asia, Madagascar. Methods To investigate the form of the density–range relationship, we regressed local density on geographical range size, and also on female body mass, because in the Primates, density correlates strongly with mass. To investigate the biological causes of the relationship, we related (1) abundance (density × range size) and (2) residuals from the density–range regression lines to various measures of (i) resource use, (ii) reproductive rate and (iii) potential specialization. All data are from the literature. Analyses were done at the level of species (n = 140), genera (n = 60) and families/subfamilies (n = 17). We present various levels of results, including for all data, after omission of outlier data, after correction for phylogenetic dependence, and after Bonferroni correction of probabilities for multiple comparisons. Results Regarding the form of the relationship, Madagascar primates are clear outliers (high densities in small ranges). Among the remaining three realms, the relation of density to range is weak or non‐existent at the level of species and genera. However, it is strong, tight and linear at the level of families/subfamilies (r² = 0.6, F_1,10 = 19, P < 0.01). Although among primates, density is very significantly related to mass, at no taxonomic level is range size related to body mass. Consequently, removing the effects of mass makes little to no difference to density–range results. Regarding the biology of the relationship, only traits indicative of specialization are associated with abundance (meaning numbers): rare taxa are more specialized than are abundant taxa. The association is largely via range size, not density. Across families, no traits correlate significantly with the density–range relationship, nor with deviations from it, despite the strength of the relationship at this taxonomic level. Main conclusions We suggest that in macroecology, analysis at taxonomic levels deeper than that of the relatively ephemeral species can be appropriate. We argue that the several purely methodological explanations for the positive density–range size relationship in primates can be rejected. Of the various biological hypotheses, those having to do with specialization–generalization seem the only applicable ones. The fact that the relationship is entirely via range size, not via density, means that while we might have a biology of range size, we do not yet have one of the density–geographical range relationship. It is probably time to search for multivariate explanations, rather than univariate ones. However, we can for the first time, for at least primates, suggest that any association of abundance or range size with specialization is via the number of different subtaxa, not the average degree of specialization of each subtaxon. The implication for conservation is obvious.     

Abortive versus productive viral infection of dendritic cells with a paramyxovirus results in differential upregulation of select costimulatory molecules

Dendritic cells are the most potent antigen-presenting cell for priming naive T cells. Optimal activation of T cells requires that dendritic cells undergo a process of maturation resulting in the increased expression of costimulatory molecules, such as CD40, CD86, and CD80, and the production of cytokines. In this study we analyzed the effect of infection of dendritic cells obtained from two strains of mice, BALB/c and C57BL/6, with the paramyxovirus simian virus 5 (SV5). Our results show that C57BL/6 bone marrow-derived dendritic cells (BMDC) are much more permissive to infection with SV5 at a multiplicity of infection (MOI) of 10 PFU/cell compared to BALB/c BMDC, as determined by the production of viral proteins and progeny. However, infection of BALB/c BMDC with a higher MOI of 50 PFU/cell resulted in a productive infection with the production of significant amounts of viral proteins and progeny. Regardless of the permissivity to infection, both BALB/c and C57BL/6 BMDC efficiently upregulated CD40 and CD86. However, CD80 upregulation correlated with the level of expression of viral proteins and the production of viral progeny. While secreted alpha/beta interferon was required for increased expression of all three molecules, optimal CD80 expression was dependent on an additional signal provided by a productive viral infection. These findings provide evidence that the signals controlling the expression of costimulatory molecules following viral infection are distinct. Further, they suggest that the amount of virus encountered and/or the permissivity of a dendritic cell to infection can alter the resulting maturation phenotype and functional capacity of the infected dendritic cell.     

Altered function in CD8+ T cells following paramyxovirus infection of the respiratory tract

For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in approximately 85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.     

Preferential apoptosis of HIV-1-specific CD4+ T cells

In contrast to other viral infections such as CMV, circulating frequencies of HIV-1-specific CD4+ T cells in peripheral blood are quantitatively diminished in the majority of HIV-1-infected individuals. One mechanism for this quantitative defect is preferential infection of HIV-1-specific CD4+ T cells, although <10% of HIV-1-specific CD4+ T cells are infected. Apoptosis has been proposed as an important contributor to the pathogenesis of CD4+ T cell depletion in HIV/AIDS. We show here that, within HIV-1-infected individuals, a greater proportion of ex vivo HIV-1-specific CD4+ T cells undergo apoptosis compared with CMV-specific CD4+ T cells (45 vs 7.4%, respectively, p < 0.05, in chronic progressors). The degree of apoptosis within HIV-1-specific CD4+ T cells correlates with viral load and disease progression, and highly active antiretroviral therapy abrogates these differences. The data support a mechanism for apoptosis in these cells similar to that found in activation-induced apoptosis through the TCR, resulting in oxygen-free radical production, mitochondrial damage, and caspase-9 activation. That HIV-1 proteins can also directly enhance activation-induced apoptosis supports a mechanism for a preferential induction of apoptosis of HIV-1-specific CD4+ T cells, which contributes to a loss of immunological control of HIV-1 replication.     

Rb is required for progression through myogenic differentiation but not maintenance of terminal differentiation

To investigate the requirement for pRb in myogenic differentiation, a floxed Rb allele was deleted either in proliferating myoblasts or after differentiation. Myf5-Cre mice, lacking pRb in myoblasts, died immediately at birth and exhibited high numbers of apoptotic nuclei and an almost complete absence of myofibers. In contrast, MCK-Cre mice, lacking pRb in differentiated fibers, were viable and exhibited a normal muscle phenotype and ability to regenerate. Induction of differentiation of Rb-deficient primary myoblasts resulted in high rates of apoptosis and a total inability to form multinucleated myotubes. Upon induction of differentiation, Rb-deficient myoblasts up-regulated myogenin, an immediate early marker of differentiation, but failed to down-regulate Pax7 and exhibited growth in low serum conditions. Primary myoblasts in which Rb was deleted after expression of differentiated MCK-Cre formed normal multinucleated myotubes that did not enter S-phase in response to serum stimulation. Therefore, Rb plays a crucial role in the switch from proliferation to differentiation rather than maintenance of the terminally differentiated state.     

A novel role for ABCA1-generated large pre-�� migrating nascent HDL in the regulation of hepatic VLDL triglyceride secretion

In Tangier disease, absence of ATP binding cassette transporter A1 (ABCA1) results in reduced plasma HDL and elevated triglyceride (TG) levels. We hypothesized that hepatocyte ABCA1 regulates VLDL TG secretion through nascent HDL production. Silencing of ABCA1 expression in oleate-stimulated rat hepatoma cells resulted in: 1) decreased large nascent HDL (>10 nm diameter) and increased small nascent HDL (<10 nm) formation, 2) increased large buoyant VLDL1 particle secretion, and 3) decreased phosphatidylinositol-3 (PI3) kinase activation. Nascent HDL-containing conditioned medium from rat hepatoma cells or HEK293 cells transfected with ABCA1 was effective in increasing PI3 kinase activation and reducing VLDL TG secretion in ABCA1-silenced hepatoma cells. Addition of isolated large nascent HDL particles to ABCA1-silenced hepatoma cells inhibited VLDL TG secretion to a greater extent than small nascent HDL. Similarly, addition of recombinant HDL, but not human plasma HDL, was effective in attenuating TG secretion and increasing PI3 kinase activation in ABCA1-silenced cells. Collectively, these data suggest that large nascent HDL particles, assembled by hepatic ABCA1, generate a PI3 kinase-mediated autocrine signal that attenuates VLDL maturation and TG secretion. This pathway may explain the elevated plasma TG concentration that occurs in most Tangier subjects and may also account, in part, for the inverse relationship between plasma HDL and TG concentrations in individuals with compromised ABCA1 function.     

Thematic review series: patient-oriented research. Recent advances in liver triacylglycerol and fatty acid metabolism using stable isotope labeling techniques

Isotopic measurement of biosynthetic rates of lipids in VLDL particles has long posed difficult technical problems. In this review, key methodologic issues and recent technical advances are discussed. A common problem for all biosynthetic measurements is the requirement to measure isotopic labeling of the true intracellular biosynthetic precursor pool. Two techniques that address this problem for lipid biosynthesis, and that are applicable to humans, have been developed-the combinatorial probability method (or mass isotopomer distribution analysis) and (2)H(2)O incorporation. The theoretical basis and practical application of these methods, both of which involve mass spectrometry, are described. Issues relevant to specific lipid components of VLDL, such as differences in the labeling of the various particle lipids (phospholipid, cholesterol, etc.), and the contribution of an intrahepatic cytosolic triacylglycerol (TG) storage pool to VLDL-TG are discussed. In summary, advances in stable isotope-mass spectrometric techniques now permit accurate measurement of liver-TG synthesis and flux. In vivo regulation of the synthesis, assembly, and secretion of VLDL-TG in humans is thereby accessible to direct investigation. Patient-oriented research in conditions such as dyslipidemia and hepatic steatosis is made feasible by these scientific advances.     

Specific sequence motifs direct the oxygenation and chlorination of tryptophan by myeloperoxidase

Most studies of protein oxidation have typically focused on the reactivity of single amino acid side chains while ignoring the potential importance of adjacent sequences in directing the reaction pathway. We previously showed that hypochlorous acid (HOCl), a specific product of myeloperoxidase, inactivates matrilysin by modifying adjacent tryptophan and glycine (WG) residues in the catalytic domain. Here, we use model peptides that mimic the region of matrilysin involved in this reaction, VVWGTA, VVWATA, and the library VVWXTA, to determine whether specific sequence motifs are targeted for chlorination or oxygenation by myeloperoxidase. Our results demonstrate that HOCl generated by myeloperoxidase or activated neutrophils converts the peptide VVWGTA to a chlorinated product, WG+32(Cl). Tandem mass spectrometry in concert with high resolution 1H and two-dimensional NMR analysis revealed that the modification required cross-linking of the tryptophan to the amide of glycine followed by chlorination of the indole ring of tryptophan. In contrast, when glycine in the peptide was replaced with alanine, the major products were mono- and dioxygenated tryptophan residues. When the peptide library VVWXTA (where X represents all 20 common amino acids) was exposed to HOCl, only WG produced a high yield of the chloroindolenine derivative. However, when glycine was replaced by other amino acids, oxygenated tryptophan derivatives were the major products. Our observations indicate that WG may represent a specific sequence motif in proteins that is targeted for chlorination by myeloperoxidase.