CD25+CD127+Foxp3- Cells Represent a Major Subpopulation of CD8+ T Cells in the Eye Chambers of Normal Mice.

The aim of this study has been to determine whether eye chambers constitute part of the normal migratory pathway of naive CD4+ and CD8+ T cells in mouse and if natural CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells are present within these eye compartments. To this aim, the cells obtained from aqueous humor (AH) of normal mice were phenotyped in terms of the expression CD4, CD8, CD25, CD127 and transcription factor Foxp3. The mean percentage of CD8+ T cells in the total AH lymphocyte population was as high as 28.69%; the mean percentage of CD8high and CD8low cells in this population was 34.09% and 65.91%, respectively. The presence of cells with the regulatory phenotype, i.e. CD25+Foxp3+ cells, constituted only 0.32% of CD8+ T cell subset. Regarding the expression of CD25, AH CD8+ T cells were an exceptional population in that nearly 85% of these cells expressed this molecule without concomitant Foxp3 expression. Despite having this phenotype, they should not be viewed as activated cells because most of them co-expressed CD127, which indicates that they are naive lymphocytes. With regard to the markers applied in the present research, CD8+CD25+CD127+Foxp3- T cells represent the most numerous subset of AH CD8+ cells. The results suggest that eye chambers in mice are an element in the normal migratory pathway of naive CD8+ T cells. The study presented herein demonstrated only trace presence of CD4+ cells in the eye chambers, as the mean percentage of these cells was just 0.56. Such selective and specific homing of CD8+ and CD4+ cells to the eye chambers is most clearly engaged in the induction and maintenance of ocular immune privilege

Development, Validation, and Application of the LC-MS/MS Method for Determination of 4-Acetamidobenzoic Acid in Pharmacokinetic Pilot Studies in Pigs

Each drug has pharmacokinetics that must be defined for the substance to be used in humans and animals. Currently, one of the basic analytical tools for pharmacokinetics studies is high-performance liquid chromatography coupled with mass spectrometry. For this analytical method to be fully reliable, it must be properly validated. Therefore, the aims of this study were to develop and validate a novel analytical method for 4-acetamidobenzoic acid, a component of the antiviral and immunostimulatory drug Inosine Pranobex, and to apply the method in the first pharmacokinetics study of 4-acetamidobenzoic acid in pigs after oral administration. Inosine Pranobex was administered under farm conditions to pigs via drinking water 2 h after morning feeding at doses of 20, 40, and 80 mg/kg. For sample preparation, we used liquid–liquid extraction with only one step—protein precipitation with 1 mL of acetonitrile. As an internal standard, we used deuterium labeled 4-acetamidobenzoic acid. The results indicate that the described method is replicable, linear (r2 ≥ 0.99), precise (2.11% to 13.81%), accurate (89% to 98.57%), selective, and sensitive (limit of quantitation = 10 ng/mL). As sample preparation requires only one step, the method is simple, effective, cheap, and rapid. The results of the pilot pharmacokinetics study indicate that the compound is quickly eliminated (elimination half-life from 0.85 to 1.42 h) and rapidly absorbed (absorption half-life from 0.36 to 2.57 h), and that its absorption increases exponentially as the dose is increased

Tree Age Effects on Fine Root Biomass and Morphology over Chronosequences of <i>Fagus sylvatica</i>, <i>Quercus robur</i> and <i>Alnus glutinosa</i> Stands

<div><p>There are few data on fine root biomass and morphology change in relation to stand age. Based on chronosequences for beech (9–140 years old), oak (11–140 years) and alder (4–76 years old) we aimed to examine how stand age affects fine root biomass and morphology. Soil cores from depths of 0–15 cm and 16–30 cm were used for the study. In contrast to previously published studies that suggested that maximum fine root biomass is reached at the canopy closure stage of stand development, we found almost linear increases of fine root biomass over stand age within the chronosequences. We did not observe any fine root biomass peak in the canopy closure stage. However, we found statistically significant increases of mean fine root biomass for the average individual tree in each chronosequence. Mean fine root biomass (0–30 cm) differed significantly among tree species chronosequences studied and was 4.32 Mg ha^-1, 3.71 Mg ha^-1 and 1.53 Mg ha^-1, for beech, oak and alder stands, respectively. The highest fine root length, surface area, volume and number of fine root tips (0–30 cm soil depth), expressed on a stand area basis, occurred in beech stands, with medium values for oak stands and the lowest for alder stands. In the alder chronosequence all these values increased with stand age, in the beech chronosequence they decreased and in the oak chronosequence they increased until ca. 50 year old stands and then reached steady-state. Our study has proved statistically significant negative relationships between stand age and specific root length (SRL) in 0–30 cm soil depth for beech and oak chronosequences. Mean SRLs for each chronosequence were not significantly different among species for either soil depth studied. The results of this study indicate high fine root plasticity. Although only limited datasets are currently available, these data have provided valuable insight into fine root biomass and morphology of beech, oak and alder stands.</p></div

The Aedileship in the Roman Republic

The search for origins of the republican aedileship presents a difficult task. At first sight the story about foundation and evolution of this magistracy lies in ancient sources. On the closer look it may be discerned, that the same sources have their own present intentions. They search for a tradition in oral based history to legitimise the current status of the aedileship and present it in historical context. Yet another question raises ambiguity. The twin character of the magistracy blurs our perceptions and makes it challenging to distinguish whether plebeian and curule aedileships are evolving intertwined or apart of each other. Nevertheless, delving upon the wide variety of ancient sources it is still possible to reconstruct the basic functions and duties of the aediles. Furthermore, the aedileship has to be looked upon in broader picture, in order to figure out, how it is situated in the system of republican magistracies and why the roman aristocrats strived to serve as aediles. The main purpose of this paper is to bring the aedileship out of the shadows and present it as full pledged research topic. Starting with aedileship it may embark us on questioning our knowledge of the republican magistracies. Powered by TCPDF (www.tcpdf.org

CD25⁺CD127⁺Foxp3^- Cells Represent a Major Subpopulation of CD8⁺ T Cells in the Eye Chambers of Normal Mice - Fig 2

<p><b>Expression/co-expression of CD25 and Foxp3 (A) and CD25 and CD127 (B) on CD8</b>^<b>+</b><b>cells in mouse peripheral blood and aqueous humor.</b> The results are expressed as a percentage of CD25⁺Foxp3⁺, CD25⁺Foxp3^-, CD25^-Foxp3⁺, CD25^-Foxp3^- (A), CD25⁺CD127⁺, CD25⁺CD127^-, CD25^-CD127⁺ and CD25^-CD127^- cells (B) within CD8⁺ T lymphocyte population. Results are the mean (± SD) of 3 independent experiments for each type of labeling. Peripheral blood samples were collected from individual mice [n = 15 (A); n = 15 (B)], whereas each aqueous humor sample consisted of cells pooled from the eyeballs of 5 (or sometimes more) mice [n = 15 samples (A); n = 15 samples (B)]. Examples of cytograms (dot plots) from different samples for different experiments (C). On the basis of expression/co-expression of CD25 and Foxp3 or CD25 and CD127, CD8⁺ T cells were subdivided into the following subsets: CD25⁺Foxp3⁺, CD25⁺Foxp3^-, CD25^-Foxp3⁺, CD25^-Foxp3^- (panels 1 and 2), CD25⁺CD127⁺, CD25⁺CD127^-, CD25^-CD127⁺ and CD25^-CD127^- cells (panels 3 and 4). Fluorescence minus one (FMO) staining was used to confirm the gating strategy used to identify CD25- (panels 1 and 3), Foxp3- (panel 1) and CD127-expressing cells (panel 3). Gated CD4⁺ T cells from peripheral blood served as a positive control for Foxp3 staining (panel 2). *P < 0.001.</p

Relationships between stand age and fine root biomass for the average individual tree in each stand (kg tree^-1) for 0–30 cm soil depth for beech, oak and alder chronosequences, modeled by Generalized Additive Models (GAM).

<p>The mean individual tree root biomass was calculated by dividing the respective fine root biomass for each stand per ha (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s004" target="_blank">S1 Table</a>) by the number of trees in the stand per ha (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.t001" target="_blank">Table 1</a>). Shaded area represents the confidence interval of GAM. Points represent mean values for each tree stand, raw datapoints are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s003" target="_blank">S1 File</a>.</p

The distribution of single- and double-positive CD4⁺ and CD8⁺ T cells in mouse peripheral blood and aqueous humor.

<p>The results are expressed as percentages of CD4⁺CD8^-, CD4^-CD8⁺ and CD4⁺CD8⁺ T cells within the total lymphocyte population (A) and as percentages of CD8^high and CD8^low cells among the CD8⁺ T cell subset (B). Results are the mean (± SD) of 6 independent experiments. Peripheral blood samples were collected from individual mice (n = 30), whereas each aqueous humor sample consisted of cells pooled from the eyeballs of 5 (or sometimes more) mice (n = 30 samples). Examples of cytograms (dot plots) from different samples for different experiments (C). As the first step, the lymphocyte population in peripheral blood was gated on the basis of forward and side scatter (FSC and SSC, respectively; panel 1 and 3). The location of this gate served as a point of reference to set the lymphocyte gate for aqueous humor samples. CD4⁺CD8^-, CD4^-CD8⁺ and CD4⁺CD8⁺ T cell subsets were defined according to the expression of CD4 and CD8 within the gated lymphocyte subpopulation (panels 2 and 4). Relative to the intensity of CD8 expression, the CD8⁺ T cell population was subdivided into CD8^high and CD8^low cell subsets (panels 2 and 4). Fluorescence minus one (FMO) staining was used to confirm the gating strategy used to identify CD8-expressing cells (panels 2 and 4). *P < 0.01, **P < 0.001.</p

Mean fine root biomass (±SE) from 0–30 cm soil depth for beech, oak and alder stand chronosequences.

<p>Analysis of variance was performed to show significance of differences among stand chronosequences for total fine root biomass. Same letters in Tukey’s test show lack of differences among mean values of fine root biomass of beech, oak and alder stands.</p

Relationships between stand age and fine root biomass expressed on a stand area basis (g m^-2) for 0–30 cm soil depth for beech, oak and alder chronosequences.

<p>Relationships were modeled using Generalized Additive Models. Shaded area represents the confidence interval of GAM. Points represent mean values for each tree stand, raw datapoints are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s003" target="_blank">S1 File</a>.</p

Relationships between stand age and fine root length, surface area, volume and number of root tips for the average individual tree in each stand for 0–30 cm soil depth for beech, oak and alder chronosequences modeled by Generalized Additive Models (GAM).

<p>The mean individual fine root indices were calculated by dividing the particular root traits values for each stand per ha (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s005" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s006" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s007" target="_blank">S4</a> Tables) by the number of trees in the stand per ha (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.t001" target="_blank">Table 1</a>). Shaded area represents the confidence interval of GAM. Points represent mean values for each tree stand, raw datapoints are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148668#pone.0148668.s003" target="_blank">S1 File</a>.</p