Rab17 and Rab18, Small GTPases with Specificity for Polarized Epithelial Cells: Genetic Mapping in the Mouse

The Rab subfamily of small GTPases plays an important role in the regulation of membrane traffic in eukaryotic cells. While most Rab proteins are equally expressed in polarized and nonpolarized cells, Rab17 and Rab18 show epithelial cell specificity. Here we report the genetic mapping of Rab17 and Rab18 on mouse chromosomes 1 and 18, respectively. We also discuss some implications ofRab17andRab18mapping, including their candidacy for the mouse mutationsln(leaden),Tw(twirler), andax(ataxia).     

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines

Background

Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment.

Methodology/Principal Findings

Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1β, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited.

Conclusions/Significance

The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.     

Using genetic techniques to quantify reinvasion,survival and in situ breeding rates during control operations

Determining the origin of individuals caught during a control/eradication programme enables conservation managers to assess the reinvasion rates of their target species and evaluate the level of success of their control methods. We examine how genetic techniques can focus management by distinguishing between hypotheses of ‘reinvasion’ and ‘survivor’, and defining kin groups for invasive stoats (Mustela erminea) on Secretary Island, New Zealand. 205 stoats caught on the island were genotyped at 16 microsatellite loci, along with 40 stoats from the opposing mainland coast, and the age and sex were determined for each individual. Using these data, we compare and combine a variety of genetic techniques including genetic clustering, population assignment and kinship‐based techniques to assess the origin of each stoat. The population history and individual movement could be described in fine detail, with results indicating that both in‐situ survival and breeding, and reinvasion are occurring. Immigration to the island was found to be generally low, apart from in 1 year where around 8 stoats emigrated from the mainland. This increased immigration was probably linked to a stoat population spike on the mainland in that year, caused by a masting event of southern beech forest (Nothofagus sp.) and the subsequent rodent irruption. Our study provides an example of some of the ways genetic analyses can feed directly into informing management practices for invasive species.     

Effect of a diabetic state on myometrial ultrastructure and isolated uterine contractions in the rat

Alterations in rat myometrial ultrastructure and in vivo uterine contractile responses to oxytocin were examined in estradiol-treated (40 micrograms/kg) euglycemic and streptozotocin-induced (85 mg/kg) diabetic rats. Myometrial morphology was examined 18, 24, and 36 hr after estradiol administration. At the time points examined, nuclei of myometrial cells from euglycemic and diabetic rats were pleomorphic and contained large areas of heterochromatin dispersed throughout the nuclei. Mitochondria were round to oval in shape and contained a dense matrix with cristae that extended across the mitochondria. Myofilaments were found in both euglycemic and diabetic cells but the relative number of myofilaments in diabetic cells appeared to be less than the number found in myometrial cells removed from euglycemic animals. The number of free cytoplasmic ribosomes in diabetic cells also appeared to be less than those found in euglycemic cells. In order to determine if apparent differences in the number of myofilament found in diabetic myometrial cells could be correlated with changes in uterine contractile responses to hormones, oxytocin dose-response curves (10(-8) to 10(-5) M) were examined in isolated uteri removed from saline-injected and estradiol-injected (24-hr pretreatments) euglycemic and diabetic rats. The maximal contractile responses (milligrams tension developed per milligrams tissue) in saline-injected euglycemic and diabetic rats were 49 +/- 5 and 36 +/- 4, respectively, while maximal contractile responses in estradiol-injected euglycemic and diabetic rats were 68 +/- 7 and 45 +/- 5, respectively. Maximal contractile responses induced by oxytocin in estradiol-treated diabetic uteri were significantly smaller than the contractile responses measured in euglycemic estradiol-treated uteri. This study demonstrates that estradiol-induced changes in both myometrial cell morphology and in vitro uterine contractile responses to oxytocin are altered in diabetic rats.     

Plant root distributions and nitrogen uptake predicted by a hypothesis of optimal root foraging

CO(2)-enrichment experiments consistently show that rooting depth increases when trees are grown at elevated CO(2) (eCO(2)), leading in some experiments to increased capture of available soil nitrogen (N) from deeper soil. However, the link between N uptake and root distributions remains poorly represented in forest ecosystem and global land-surface models. Here, this link is modeled and analyzed using a new optimization hypothesis (MaxNup) for root foraging in relation to the spatial variability of soil N, according to which a given total root mass is distributed vertically in order to maximize annual N uptake. MaxNup leads to analytical predictions for the optimal vertical profile of root biomass, maximum rooting depth, and N-uptake fraction (i.e., the proportion of plant-available soil N taken up annually by roots). We use these predictions to gain new insight into the behavior of the N-uptake fraction in trees growing at the Oak Ridge National Laboratory free-air CO(2)-enrichment experiment. We also compare MaxNup with empirical equations previously fitted to root-distribution data from all the world's plant biomes, and find that the empirical equations underestimate the capacity of root systems to take up N.     

Effects of elevated [CO2] on forest growth and carbon storage: a modelling analysis of the consequences of changes in litter quality/quantity and root exudation

Many researchers have proposed that the stimulus of plant growth under elevated [CO₂] observed in short-term experiments will be moderated in the longer term by a reduction in soil nitrogen (N) availability linked to decreased litter quality and/or increased litter production. However, these negative feedbacks may be offset to some extent by a stimulus in N fixation linked to increased root exudation. The aim of this modelling study is to examine how changes in litter quality/quantity and root exudation –- if they occur –- will affect the CO₂ responses of net primary productivity and ecosystem carbon (C) storage on different timescales. We apply a model of C and N cycling in forest ecosystems (G’DAY) to stands of Norway spruce (Picea abies, L. Cast) growing at a N-limited experimental site at Flakaliden, Sweden, and draw the following conclusions: (1) in the absence of changes in litter quality and root exudation, the short-term CO₂ stimulus of litter quantity leads to only a minimal CO₂ stimulus of productivity or C storage in the medium term (≈ 20 years) and long term (≈ 200 years), because of constraints on soil N availability; (2) increasing plant nitrogen use efficiency (via a decrease in the N:C ratio of new litter) makes little impact on these results; (3) a significant CO₂ response in the medium term requires a substantial decrease in the N:C ratio of older litter, when it is approaching stabilisation as soil organic matter, although the long-term CO₂ response remains small; and (4) an increase in N fixation leads to a small effect on productivity in the short term, but a very large effect on both productivity and C storage in the long term. These results suggest that soil N constraints on the long-term CO₂-fertilisation effect can be overcome to a significant extent only by increases in N acquisition, although only modest increases may be required. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tumor-infiltrating T cells correlate with NY-ESO-1-specific autoantibodies in ovarian cancer

Background

Tumor-infiltrating CD8+ T cells are correlated with prolonged progression-free and overall survival in epithelial ovarian cancer (EOC). A significant fraction of EOC patients mount autoantibody responses to various tumor antigens, however the relationship between autoantibodies and tumor-infiltrating T cells has not been investigated in EOC or any other human cancer. We hypothesized that autoantibody and T cell responses may be correlated in EOC and directed toward the same antigens.

Methodology and Principal Findings

We obtained matched serum and tumor tissue from 35 patients with high-grade serous ovarian cancer. Serum samples were assessed by ELISA for autoantibodies to the common tumor antigen NY-ESO-1. Tumor tissue was examined by immunohistochemistry for expression of NY-ESO-1, various T cell markers (CD3, CD4, CD8, CD25, FoxP3, TIA-1 and Granzyme B) and other immunological markers (CD20, MHC class I and MHC class II). Lymphocytic infiltrates varied widely among tumors and included cells positive for CD3, CD8, TIA-1, CD25, FoxP3 and CD4. Twenty-six percent (9/35) of patients demonstrated serum IgG autoantibodies to NY-ESO-1, which were positively correlated with expression of NY-ESO-1 antigen by tumor cells (r = 0.57, p = 0.0004). Autoantibodies to NY-ESO-1 were associated with increased tumor-infiltrating CD8+, CD4+ and FoxP3+ cells. In an individual HLA-A2+ patient with autoantibodies to NY-ESO-1, CD8+ T cells isolated from solid tumor and ascites were reactive to NY-ESO-1 by IFN-γ ELISPOT and MHC class I pentamer staining.

Conclusion and Significance

We demonstrate that tumor-specific autoantibodies and tumor-infiltrating T cells are correlated in human cancer and can be directed against the same target antigen. This implies that autoantibodies may collaborate with tumor-infiltrating T cells to influence clinical outcomes in EOC. Furthermore, serological screening methods may prove useful for identifying clinically relevant T cell antigens for immunotherapy.     

The effects of mice on stoats in southern beech forests

Introduced stoats (Mustela erminea) are important invasive predators in southern beech (Nothofagus sp.) forests in New Zealand. In these forests, one of their primary prey species – introduced house mice (Mus musculus), fluctuate dramatically between years, driven by the irregular heavy seed‐fall (masting) of the beech trees. We examined the effects of mice on stoats in this system by comparing the weights, age structure and population densities of stoats caught on two large islands in Fiordland, New Zealand – one that has mice (Resolution Island) and one that does not (Secretary Island). On Resolution Island, the stoat population showed a history of recruitment spikes and troughs linked to beech masting, whereas the Secretary Island population had more constant recruitment, indicating that rodents are probably the primary cause for the ‘boom and bust’ population cycle of stoats in beech forests. Resolutions Island stoats were 10% heavier on average than Secretary Island stoats, supporting the hypothesis that the availability of larger prey (mice verses wētā) leads to larger stoats. Beech masting years on this island were also correlated with a higher weight for stoats born in the year of the masting event. The detailed demographic information on the stoat populations of these two islands supports previously suggested interactions among mice, stoats and beech masting. These interactions may have important consequences for the endemic species that interact with fluctuating populations of mice and stoats.