Effects of acquired immunity and mating strategy on the genetic structure of parasite populations

Although it may have profound effects on how researchers seek to tackle many infectious diseases, little is known of the genetic structure of many pathogen populations. Previous models have suggested that if levels of cross-protection are high, parasite populations may be structured into discrete strains with nonoverlapping antigenic repertoires, even among populations that reproduce sexually. Here, I consider a discrete model of the coevolution of parasites with host-acquired immunity. In this model, if the effective recombination rate is low, strain structure can be maintained for very high levels of cross-protection. However, if the effective recombination rate is higher, this strain structure can no longer be maintained. The effective recombination rate is affected by the actual recombination rate between immunologically selected loci, the proportion of individuals that reproduce sexually, whether recombination occurs inside or outside of the host or vector, and the level of cross-protection. The model predicts that for Plasmodium falciparum, where reproduction occurs inside of a vector, we expect to see strain structure in areas of low transmission but not in areas of high transmission. Strain structure is unlikely to be seen in parasites that reproduce outside of a host or vector, such as Strongyloides ratti.     

Characterization of early,chitin-induced gene expression in Arabidopsis

Three genes (i.e., a zinc finger protein, a lectin-like protein, and AtMPK3), previously shown to respond to chitin elicitation in microarray experiments, were used to examine the response of Arabidopsis spp. to chitin addition. Maximum induction for all three genes was found upon addition of crab-shell chitin at 100 mg per liter. Threefold induction was found with a chitin concentration as low as 10(-4) mg per liter. The specificity of this response was examined using purified chitin oligomers (degree of polymerization = 2 to 8). The larger chitin oligomers (hexamer to octamer), were most effective in inducing expression of the three genes assayed. Gene induction was observed after the addition of 1 nM chitin octamer. The protein kinase inhibitors staurosporine and K252a effectively suppressed chitin-induced gene expression, while the protein phosphatase inhibitors calyculin A and okadaic acid induced the accumulation of mRNA in the absence of chitin. The phosphorylation event necessary for transmission of the chitin signal was completed within the first 20 min of chitin addition. The level of chitin-induced gene expression of the lectin-like protein and AtMPK3 was not significantly changed in mutants blocked in the jasmonic acid (JA, jar1)-, ethylene (ein2)-, or salicylic acid (SA, pad4, npr1, and eds5)-dependent pathway. In contrast, expression of mRNA for the zinc finger protein was reduced in the mutants affected in the JA- or SA-dependent pathway.     

Production and characterisation of monoclonal antibodies to ovine interleukin-5

Monoclonal antibodies (MAbs) were developed against recombinant ovine interleukin-5 (IL-5) produced in the baculovirus expression vector system. One MAb, D11 (isotype IgG1), neutralised the activity of both recombinant and native sources of IL-5 in a biological assay (Baf cell assay) but was only weakly reactive in immunocytochemistry. Conversely, a second MAb, A8 (isotype IgA), successfully detected IL-5 in immunocytochemistry but did not display neutralising activity. The development of these MAbs will enable the assay of ovine IL-5 in vitro and permit studies into the role of hypersensitivity reactions in sheep by neutralisation of IL-5 in vivo.     

Longitudinal variance components models for systolic blood pressure, fitted using Gibbs sampling

This paper describes an analysis of systolic blood pressure (SBP) in the Genetic Analysis Workshop 13 (GAW13) simulated data. The main aim was to assess evidence for both general and specific genetic effects on the baseline blood pressure and on the rate of change (slope) of blood pressure with time. Generalized linear mixed models were fitted using Gibbs sampling in WinBUGS, and the additive polygenic random effects estimated using these models were then used as continuous phenotypes in a variance components linkage analysis. The first-stage analysis provided evidence for general genetic effects on both the baseline and slope of blood pressure, and the linkage analysis found evidence of several genes, again for both baseline and slope.     

Genome-wide linkage analysis of longitudinal phenotypes using σ<Superscript>2</Superscript><Subscript>A</Subscript> random effects (SSARs) fitted by Gibbs sampling

The study of change in intermediate phenotypes over time is important in genetics. In this paper we explore a new approach to phenotype definition in the genetic analysis of longitudinal phenotypes. We utilized data from the longitudinal Framingham Heart Study Family Cohort to investigate the familial aggregation and evidence for linkage to change in systolic blood pressure (SBP) over time. We used Gibbs sampling to derive sigma-squared-A-random-effects (SSARs) for the longitudinal phenotype, and then used these as a new phenotype in subsequent genome-wide linkage analyses. Additive genetic effects (sigma2A.time) were estimated to account for approximately 9.2% of the variance in the rate of change of SBP with age, while additive genetic effects (sigma2A) were estimated to account for approximately 43.9% of the variance in SBP at the mean age. The linkage results suggested that one or more major loci regulating change in SBP over time may localize to chromosomes 2, 3, 4, 6, 10, 11, 17, and 19. The results also suggested that one or more major loci regulating level of SBP may localize to chromosomes 3, 8, and 14. Our results support a genetic component to both SBP and change in SBP with age, and are consistent with a complex, multifactorial susceptibility to the development of hypertension. The use of SSARs derived from quantitative traits as input to a conventional linkage analysis appears to be valuable in the linkage analysis of genetically complex traits. We have now demonstrated in this paper the use of SSARs in the context of longitudinal family data.     

Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin

The skin accommodates multiple dendritic cell (DC) subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal) and chicken ovalbumin (OVA) under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin⁺ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC) as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin⁺ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.     

Collective cell migration of smooth muscle and endothelial cells: impact of injury versus non-injury stimuli

Background

Cell migration is a vital process for growth and repair. In vitro migration assays, utilized to study cell migration, often rely on physical scraping of a cell monolayer to induce cell migration. The physical act of scrape injury results in numerous factors stimulating cell migration – some injury-related, some solely due to gap creation and loss of contact inhibition. Eliminating the effects of cell injury would be useful to examine the relative contribution of injury versus other mechanisms to cell migration. Cell exclusion assays can tease out the effects of injury and have become a new avenue for migration studies. Here, we developed two simple non-injury techniques for cell exclusion: 1) a Pyrex® cylinder - for outward migration of cells and 2) a polydimethylsiloxane (PDMS) insert - for inward migration of cells. Utilizing these assays smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs) migratory behavior was studied on both polystyrene and gelatin-coated surfaces.

Results

Differences in migratory behavior could be detected for both smooth muscle cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1.26 % per hour and 1.59 % per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05 % per hour. The slowest migration occurred with the same conditions but on a polystyrene surface at a rate of 0.33 % per hour.

Conclusion

For SMCs, injury is a dominating factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays - the in-growth and out-growth assays, provide a means to determine pure migratory behavior of cells in comparison to migration confounded by cell wounding and injury.

     

Detection and Characterization of Clade 1 Reassortant H5N1 Viruses Isolated from Human Cases in Vietnam during 2013

Highly pathogenic avian influenza (HPAI) H5N1 is endemic in Vietnamese poultry and has caused sporadic human infection in Vietnam since 2003. Human infections with HPAI H5N1 are of concern due to a high mortality rate and the potential for the emergence of pandemic viruses with sustained human-to-human transmission. Viruses isolated from humans in southern Vietnam have been classified as clade 1 with a single genome constellation (VN3) since their earliest detection in 2003. This is consistent with detection of this clade/genotype in poultry viruses endemic to the Mekong River Delta and surrounding regions. Comparison of H5N1 viruses detected in humans from southern Vietnamese provinces during 2012 and 2013 revealed the emergence of a 2013 reassortant virus with clade 1.1.2 hemagglutinin (HA) and neuraminidase (NA) surface protein genes but internal genes derived from clade 2.3.2.1a viruses (A/Hubei/1/2010-like; VN12). Closer analysis revealed mutations in multiple genes of this novel genotype (referred to as VN49) previously associated with increased virulence in animal models and other markers of adaptation to mammalian hosts. Despite the changes identified between the 2012 and 2013 genotypes analyzed, their virulence in a ferret model was similar. Antigenically, the 2013 viruses were less cross-reactive with ferret antiserum produced to the clade 1 progenitor virus, A/Vietnam/1203/2004, but reacted with antiserum produced against a new clade 1.1.2 WHO candidate vaccine virus (A/Cambodia/W0526301/2012) with comparable hemagglutination inhibition titers as the homologous antigen. Together, these results indicate changes to both surface and internal protein genes of H5N1 viruses circulating in southern Vietnam compared to 2012 and earlier viruses.