Association of Kaposi's sarcoma-associated herpesvirus (KSHV) DNA in bronchoalveolar lavage fluid of HIV-infected persons with bronchoscopically diagnosed tracheobronchial Kaposi's sarcoma

The satisfiability problem of hybrid logics with the downarrow binder is known to be undecidable. This initiated a research program on decidable and tractable fragments. In this paper, we investigate the effect of restricting the propositional part of the language on decidability and on the complexity of the satisfiability problem over arbitrary, transitive, total frames, and frames based on equivalence relations. We also consider different sets of modal and hybrid operators. We trace the border of decidability and give the precise complexity of most fragments, in particular for all fragments including negation. For the monotone fragments, we are able to distinguish the easy from the hard cases, depending on the allowed set of operators

A TNFR2-Agonist Facilitates High Purity Expansion of Human Low Purity Treg Cells

<div><p>Regulatory T cells (Treg) are important for immune homeostasis and are considered of great interest for immunotherapy. The paucity of Treg numbers requires the need for <i>ex vivo</i> expansion. Although therapeutic Treg flow-sorting is feasible, most centers aiming at Treg-based therapy focus on magnetic bead isolation of CD4+CD25+ Treg using a good manufacturing practice compliant closed system that achieves lower levels of cell purity. Polyclonal Treg expansion protocols commonly use anti-CD3 plus anti-CD28 monoclonal antibody (mAb) stimulation in the presence of rhIL-2, with or without rapamycin. However, the resultant Treg population is often heterogeneous and pro-inflammatory cytokines like IFNγ and IL-17A can be produced. Hence, it is crucial to search for expansion protocols that not only maximize <i>ex vivo</i> Treg proliferative rates, but also maintain Treg stability and preserve their suppressive function. Here, we show that <i>ex vivo</i> expansion of low purity magnetic bead isolated Treg in the presence of a TNFR2 agonist mAb (TNFR2-agonist) together with rapamycin, results in a homogenous stable suppressive Treg population that expresses FOXP3 and Helios, shows low expression of CD127 and hypo-methylation of the <i>FOXP3</i> gene. These cells reveal a low IL-17A and IFNγ producing potential and hardly express the chemokine receptors CCR6, CCR7 and CXCR3. Restimulation of cells in a pro-inflammatory environment did not break the stability of this Treg population. In a preclinical humanized mouse model, the TNFR2-agonist plus rapamycin expanded Treg suppressed inflammation <i>in vivo</i>. Importantly, this Treg expansion protocol enables the use of less pure, but more easily obtainable cell fractions, as similar outcomes were observed using either FACS-sorted or MACS-isolated Treg. Therefore, this protocol is of great interest for the <i>ex vivo</i> expansion of Treg for clinical immunotherapy.</p></div

TNFR2-agonist facilitates <i>ex vivo</i> expansion of low purity MACS-isolated human Treg.

<p>(A) Schematic overview of low purity MACS-isolated Treg expansion strategy as described in Materials and Methods. (B) Dot plots showing a representative FOXP3 expression pattern after MACS isolation of Treg, as shown in the cumulative data graph (N = 10, right panel). (C) Cumulative graph showing fold expansion of low purity Treg that were stimulated under the conditions as indicated on the X-axis (N = 5). A Friedman test was used for comparison of three groups. (D) Flow cytometry of surface expression of HLA-DR, and intracellular expression of FOXP3 and Helios of MACS-isolated Treg before (input) and after expansion under the indicated conditions. Numbers within the quadrant gates show the percentage of positive cells. Cumulative data of %FOXP3+Helios+, %FOXP3+HLA-DR+, the median fluorescence intensity (MFI) of Helios and HLA-DR are shown in the right panel, respectively. Wilcoxon paired t-Test and Kruskal-Wallis test were used for comparison between two and multiple groups, respectively. Asterisks indicate significant differences. Rap: rapamycin; Agonist: TNFR2-agonist.</p

TNFR2-agonist preserves the stability of low purity MACS-isolated Treg during <i>ex vivo</i> expansion.

<p>Low purity MACS-isolated human Treg were cultured as described under Materials and Methods. Thereafter, the expanded Treg were harvested, washed, and analyzed for their suppressor capacity in a CFSE-based co-culture suppression assay. ^CtrlTreg, ^RapTreg and ^R/TTreg represent cells expanded in the absence or presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (A) Histograms showing the inhibition of proliferation of Tresp following the addition of graded doses of Treg. The ratios of Treg:Tresp are indicated on the top. Numbers in the histograms show the percentage of divided cells. Cumulative data (N = 6) are shown in the right panel. (B) Flow cytometry of intracellular IL-17A and IFNγ of Treg at the start of the culture (day 0) and after expansion under the indicated conditions. Dot plots showing representative data of N = 4–7 individuals as shown in the cumulative data graph (right panel). Numbers show the percentage of positive cells. Each line represents Treg derived from a specific donor were expanded under the conditions described on the X-axis. (C) Expression of CXCR3, CCR5, CCR6, CCR7, CD62L and CD27 before (day 0) and after expansion (day 16). Numbers show the percentage of positive cells. (D) Bisulphite sequencing of the TSDR of expanded Treg. Each dot represents a single experiment. (E) Expanded Treg were harvested, rested overnight, and then restimulated with anti-CD3/anti-CD28 beads in a 1:2 ratio of beads to cells, in the absence or presence of IL-1β (50 ng/mL) and IL-23 (50 ng/mL) for 2-days. Exogenous rhIL-2 (200 U/mL) was included in the cell cultures. Thereafter, intracellular production of IL-17A was analyzed using flow cytometry. Cumulative data derived from seven different donors are shown. A Friedman plus Dunns post hoc test (A, D, and E) or Kruskal-Wallis plus Dunns post hoc test (B) were used for comparison among multiple groups. Asterisks indicate significant differences. Rap: rapamycin; Agonist: TNFR2-agonist.</p

TNFR2-agonist plus rapamycin expanded FACS-sorted Treg reveal a high suppressive capacity and less IL-17A and IFNγ producing potential.

<p>High purity FACS-sorted human Treg were stimulated as described in Material and Methods. At day 7 of the cell cultures, expanded Treg were harvested, washed, and analyzed for their suppressive capacity in a CFSE-based co-culture suppression assay. ^CtrlTreg, ^RapTreg and ^R/TTreg represent cells expanded in the absence or presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (A) Histograms showing the inhibition of proliferation of responder cells following the addition of graded doses of Treg. The ratio of Treg:Tconv is indicated on the top. Numbers show the percentage of divided cells. (B) Flow cytometry of intracellular IL-17A and IFNγ of Treg before (day 0) and after expansion under the indicated conditions. Data derived from two different healthy donors are shown. Numbers show the percentage of positive cells. Rap: rapamycin; Agonist: TNFR2-agonist.</p

TNFR2-agonist plus rapamycin expanded Treg inhibit inflammation in a humanised mouse model.

<p>(A) Schematic overview of the humanised skin inflammation mouse model used. In brief, SCID mice were transplanted with a human skin graft, 21 days after engraftment, PBS (as a control), or allogeneic human PBMC (huPBMC) only or huPBMC plus Treg of interest (at a ratio of 1:1) were injected intra peritoneally. 26 days later the animals were sacrificed to analyze the mouse spleen and human skin grafts. ^RapTreg and ^R/TTreg refer to low purity MACS-isolated Treg expanded for 16-days in the presence of rapamycin-only or TNFR2-agonist plus rapamycin, respectively. (B) Representative photographs of spleens derived from mice infused with PBS, huPBMC only, or huPBMC plus Treg of interest, 21 days after the skin transplantation. Cumulative data showing the weight of spleens derived from N = 4 mice (right panel; n.d. = not determined). (C) Representative photographs showing histology (HE staining) of human skin grafts. Left panel: 10 x magnification. Right panel shows the epidermal thickness (μm) of human skin grafts following infusion of PBS, huPBMC, huPBMC plus ^RapTreg or huPBMC plus ^R/TTreg. Mean ± SD, N = 6. (D) Immunohistochemistry of human CD3+ (brown) T cell infiltration in the human dermis. A representative photograph of N = 4 as presented in the cumulative data graph is shown (right panel; Mean ± SD). 20 x magnification. Wilcoxon paried t Test was used to compare the group of mice infused with huPBMC only with other groups of mice infused with huPBMC plus Treg of interest. Asterisks indicate significant differences.</p

Maximizing the reliability of genomic selection by optimizing the calibration set of reference individuals: comparison of methods in two diverse groups of maize inbreds (Zea mays L.)

Chantier qualité GAGenomic selection refers to the use of genotypic information for predicting breeding values of selection candidates. A prediction formula is calibrated with the genotypes and phenotypes of reference individuals constituting the calibration set. The size and the composition of this set are essential parameters affecting the prediction reliabilities. The objective of this study was to maximize reliabilities by optimizing the calibration set. Different criteria based on the diversity or on the prediction error variance (PEV) derived from the realized additive relationship matrix-best linear unbiased predictions model (RA-BLUP) were used to select the reference individuals. For the latter, we considered the mean of the PEV of the contrasts between each selection candidate and the mean of the population (PEVmean) and the mean of the expected reliabilities of the same contrasts (CDmean). These criteria were tested with phenotypic data collected on two diversity panels of maize (Zea mays L.) genotyped with a 50k SNPs array. In the two panels, samples chosen based on CDmean gave higher reliabilities than random samples for various calibration set sizes. CDmean also appeared superior to PEVmean, which can be explained by the fact that it takes into account the reduction of variance due to the relatedness between individuals. Selected samples were close to optimality for a wide range of trait heritabilities, which suggests that the strategy presented here can efficiently sample subsets in panels of inbred lines. A script to optimize reference samples based on CDmean is available on request

Intraspecific variation of recombination rate in maize

Abstract Background In sexually reproducing organisms, meiotic crossovers ensure the proper segregation of chromosomes and contribute to genetic diversity by shuffling allelic combinations. Such genetic reassortment is exploited in breeding to combine favorable alleles, and in genetic research to identify genetic factors underlying traits of interest via linkage or association-based approaches. Crossover numbers and distributions along chromosomes vary between species, but little is known about their intraspecies variation. Results Here, we report on the variation of recombination rates between 22 European maize inbred lines that belong to the Dent and Flint gene pools. We genotype 23 doubled-haploid populations derived from crosses between these lines with a 50 k-SNP array and construct high-density genetic maps, showing good correspondence with the maize B73 genome sequence assembly. By aligning each genetic map to the B73 sequence, we obtain the recombination rates along chromosomes specific to each population. We identify significant differences in recombination rates at the genome-wide, chromosome, and intrachromosomal levels between populations, as well as significant variation for genome-wide recombination rates among maize lines. Crossover interference analysis using a two-pathway modeling framework reveals a negative association between recombination rate and interference strength. Conclusions To our knowledge, the present work provides the most comprehensive study on intraspecific variation of recombination rates and crossover interference strength in eukaryotes. Differences found in recombination rates will allow for selection of high or low recombining lines in crossing programs. Our methodology should pave the way for precise identification of genes controlling recombination rates in maize and other organisms.Results have been achieved in the framework of the Transnational (Germany, France, Spain) Cooperation within the PLANT-KBBE Initiative Cornfed, additionally supported by the project AMAIZING. The work was financed by grants from Agence Nationale de la Recherche ('ANR') to AC, MF, MM, and PF, grants from the Ministry of Science and Innovation (Ministerio de Ciencia e Innovación ('MICINN')) to JMG and PR, and grants from the Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, 'BMBF') to TA, AEM, MO, and CCS.Peer Reviewe