NK cell adoptive transfer combined with Ontak-mediated regulatory T cell elimination induces effective adaptive antitumor immune responses

Previous work from our laboratory showed that hydrocortisone (HC) combined with IL-15 induces expansion of activated human NK cells. We set up an experimental tumor model to evaluate the use of adoptively transferred, HC plus IL-15 (HC/IL-15)-activated and -expanded murine NK cells in the treatment of syngeneic mice carrying established lung metastases of the CT26 transplantable tumor. We also examined the effect of denileukin diftitox (Ontak) on the depletion of regulatory T cells to enhance the in vivo antitumor immunity induced by the adoptively transferred NK cells. Our results clearly demonstrate that murine DX5(+) NK cells are largely expanded in the presence of IL-15 plus HC while retaining intact their functional status. Moreover, when intravenously infused, they mediated significant antitumor responses against CT26 lung tumors in syngeneic BALB/c animals that were further enhanced upon pretreatment of the tumor-bearing animals with Ontak. Total splenocytes and isolated splenic T cells from NK-treated mice responded in vitro against CT26 tumor cells as evidenced by IFN-γ-based ELISPOT, proliferation, and cytotoxicity assays. Importantly, animals treated with Ontak plus adoptive transfer of HC/IL-15-expanded NK cells significantly retarded CT26 tumor growth after a rechallenge with the same tumor s.c. in their flanks. Taken altogether, our data suggest that NK cell adoptive transfer can trigger adaptive antitumor T cell responses, and regulatory T cell depletion by Ontak is mandatory for enabling HC/IL-15-activated NK cells to promote long-lasting adaptive antitumor immunity.     

Modeling of angioadaptation: insights for vascular development

Vascular beds are generated by vasculogenesis and sprouting angiogenesis, and these processes have strong stochastic components. As a result, vascular patterns exhibit significant heterogeneity with respect to the topological arrangement of the individual vessel segments and the characteristics (length, number of segments) of different arterio-venous pathways. This structural heterogeneity tends to cause heterogeneous distributions of flow and oxygen availability in tissue. However, these quantities must be maintained within tolerable ranges to allow normal tissue function. This is achieved largely through adjustment of vascular flow resistance by control of vessel diameters. While short-term diameter control by changes in vascular tone in arterioles and small arteries plays an important role, in the long term an even more important role is played by structural adaptation (angioadaptation), occurring in response to metabolic and hemodynamic signals. The effectiveness, stability and robustness of this angioadaptation depend sensitively on the nature and strength of the vascular responses involved and their interactions with the network structure. Mathematical models are helpful in understanding these complex interactions, and can be used to simulate the consequences of failures in sensing or signal transmission mechanisms. For the tumor microcirculation, this strategy of combining experimental observations with theoretical models, has led to the hypothesis that dysfunctional information transport via vascular connexins is a major cause of the observed vascular pathology and increased heterogeneity in oxygen distribution.     

Rejection of brood‐parasitic shiny cowbird Molothrus bonariensis nestlings by the firewood‐gatherer Anumbius annumbi?

Costs imposed by brood parasitic birds exert strong selection on their hosts to avoid parasitism. While egg rejection is a common defence, nestling rejection is rarer and less well understood. Theoretical models suggest that among non‐evicting parasites such as cowbirds nestling rejection can only evolve when levels of parasitism are high. Here we describe a possible case of early rejection of cowbird nestlings, by an infrequently parasitised host, the firewood‐gatherer Anumbius annumbi. Firewood‐gatherers accepted most shiny cowbird Molothrus bonariensis eggs despite clear differences in coloration. Cowbird eggs usually hatched 4–5 d before host eggs. All parasitic nestlings died within 48 h, and hosts continued their breeding attempts. Nestling death was most likely due to neglect since little food was found in the stomach of dead nestlings. Feeding neglect could be due to differences in visual or acoustic appearance between host and parasite hatchlings. Alternatively, hosts may refrain from feeding nestlings that hatch too early compared to their normal incubation time. At the moment our data do not allow distinction between active nestling recognition or cowbird nestling failure due to the unsuitability of the firewood‐gatherer as a host (i.e. too long incubation). Experiments are needed to tease these alternatives apart.     

Detection of monochlorobenzene metabolizing bacteria under anoxic conditions by DNA-stable isotope probing

Cultivation-independent analyses were applied to study the structural diversity of the bacterial community which developed in groundwater inoculated microcosms actively metabolizing monochlorobenzene (MCB) under anaerobic conditions. Addition of ¹³C-labelled MCB demonstrated that the community produced ¹³CO₂ as a metabolite at slightly increasing rates over a period of 1,051 days while no ¹³C-methane evolved. Genetic profiles of partial 16S rRNA genes generated with the single-strand conformation polymorphism (SSCP) technique by PCR from directly extracted total DNA revealed that, despite the long incubation period, six replicate microcosms were characterized by almost the same microbial members. Nine distinguishable contributors to the SSCP-profiles were characterized by DNA sequencing, revealing the presence of different members from the phyla Proteobacteria, Fibrobacteres and from the candidate division OD1. DNA-stable isotope probing (SIP) was applied to distinguish the actual MCB metabolizing bacteria from the other community members. This study reveals for the first time the structural diversity of an anaerobic MCB metabolizing bacterial community. However, it also demonstrates the limitations of SIP to detect bacteria slowly metabolizing carbon sources under anaerobic conditions.     

Novel estrogen-related genes and potential biomarkers of ovarian endometriosis identified by differential expression analysis

In the search for novel biomarkers of endometriosis, we selected 152 genes from the GeneLogic database based on results of genome-wide expression analysis of ovarian endometriosis, plus 20 genes related to estrogen metabolism and action. We then performed low-density array analysis of these 172 genes on 11 ovarian endometriosis samples and 9 control endometrium samples. Principal component analysis of the gene expression levels showed clear separation between the endometriosis and control groups. We identified 78 genes as differentially expressed. Based on Ingenuity pathway analysis, these differentially expressed genes were arranged into groups according to biological function. These analyses revealed that 32 differentially expressed genes are estrogen related, 23 of which have not been reported previously in connection with endometriosis. Functional annotation showed that 25 and 22 genes are associated with the biological terms "secreted" and "extracellular region", respectively. Differential expression of 4 out of 5 genes related to estrogen metabolism and action (ESR1, ESR2, PGR and BGN) was also confirmed by immunohistochemistry. Our study thus reveals differential expression of several genes that have not previously been associated with endometriosis and that encode potential novel biomarkers and drug targets.     

Analysis of short-term changes in the Arabidopsis thaliana glycerolipidome in response to temperature and light

Although the influence of temperature, particularly cold, on lipid metabolism is well established, previous studies have focused on long-term responses and have largely ignored the influence of other interacting environmental factors. Here, we present a time-resolved analysis of the early responses of the glycerolipidome of Arabidopsis thaliana plants exposed to various temperatures (4, 21 and 32°C) and light intensities (darkness, 75, 150 and 400 μmol m(-2) s(-1)), including selected combinations. Using a UPLC/MS-based lipidomic platform, we reproducibly measured most glycerolipid species reported for Arabidopsis leaves, including the classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). In addition to known lipids, we have identified previously unobserved compounds, such as 36-C PGs and eukaryotic phospholipids containing 16:3 acyl chains. Occurrence of these lipid species implies the action of new biochemical mechanisms. Exposition of Arabidopsis plants to various light and temperature regimes results in two major effects. The first is the dependence of the saturation level of PC and MGDG pools on light intensity, likely arising from light regulation of de novo fatty acid synthesis. The second concerns an immediate decrease in unsaturated species of PG at high-temperature conditions (32°C), which could mark the first stages of adaptation to heat-stress conditions. Observed changes are discussed in the context of current knowledge, and new hypotheses have been formulated concerning the early stages of the plant response to changing light and temperature conditions.     

tRNA 3′ processing in yeast involves tRNase Z,Rex1, and Rrp6

Mature tRNA 3′ ends in the yeast Saccharomyces cerevisiae are generated by two pathways: endonucleolytic and exonucleolytic. Although two exonucleases, Rex1 and Rrp6, have been shown to be responsible for the exonucleolytic trimming, the identity of the endonuclease has been inferred from other systems but not confirmed in vivo. Here, we show that the yeast tRNA 3′ endonuclease tRNase Z, Trz1, is catalyzing endonucleolytic tRNA 3′ processing. The majority of analyzed tRNAs utilize both pathways, with a preference for the endonucleolytic one. However, 3′-end processing of precursors with long 3′ trailers depends to a greater extent on Trz1. In addition to its function in the nucleus, Trz1 processes the 3′ ends of mitochondrial tRNAs, contributing to the general RNA metabolism in this organelle.