ライフサイクルとヒューマンケア: 高齢者への健康支援(平成23年度教養コア科目) 授業資料ナビゲータ(PathFinder)

担当教員:黒田久美子,野地有子,今村恵美子,永野みどり平成23年度(2011)教養コア科目授業B(こころと発達),授業コード:G14B1410

vTR-TERT interaction is not required for efficient tumor dissemination or for telomerase activity in tumor cells.

<p>A) Dissemination pattern of vP6.1mut (22 chickens) and vP6.1rev (20 chickens) for <i>in vivo</i> experiment 2. Moribund chickens were euthanized, necropsied and evaluated for lymphoma dissemination. Results are shown as percentage of animals with 1–2, 3–4 or 5–6 organs containing lymphomatous lesions. B) Mean number of tumors per animal with standard deviations. The mean number was significantly increased in the P6.1mut group indicated by the asterisk (p = 0.0381). C–D) Telomerase activity in primary tumor cells (C) and clonal LCLs (D) derived from vRB-1B or vP6.1mut infected animals as indicated using the Cy5 gel based TRAP-assay. TRAP products and the internal control (IC) are indicated. E–F) Quantification of telomerase activity in primary tumor cells (E) and established LCLs (F). The data are shown as mean telomerase activity relative to the positive control in three (E) or two (F) independent experiments.</p

MDV genome organization and P6.1 stem-loop mutation.

<p>A) Schematic representation of the MDV genome including the unique-short and -long regions (U_S, U_L) flanked by terminal and internal repeat regions (TR_S, TR_L, IR_S, IR_L). The focus on the vTR containing regions shows the telomeric repeat region present in the MDV genome (left), vTR including its conserved regions (CR) 1–8, and the three exons of the neighboring vIL-8 gene (right). B) Restriction fragment length polymorphism analyses of pRB-1B (lane 2, 6, 10), vP6.1mut (lane 3, 7, 11) and vP6.1rev (lane 4, 8, 12) using the indicated restriction enzymes. Lane 1, 5, and 9 show the 1 kb plus ladder (Invitrogen) ranging from 2 kbp (lowest band) till 12 kbp (highest band) in exact 1 kbp increments.</p

P6.1 stem-loop mutation does not affect lytic replication <i>in vivo</i>, but delays MD incidence.

<p>A) qPCR analysis of the viral <i>ICP4</i> gene and the host <i>iNOS</i> gene. Blood samples were taken at 4, 7, 10, 14, 21, and 28 dpi and total DNA was extracted. Mean MDV genome copies/10⁶ blood cells of eight infected chickens per group as determined by qPCR analysis are shown with standard deviations (error bars). B) 1^st animal experiment: MD incidence in percent in chickens infected with vRB-1B (n = 5), vP6.1mut (n = 10) and vP6.1rev (n = 8) during the indicated time period C) 2^nd animal experiment: MD incidence in percent of vP6.1mut (n = 22) and vP6.1rev (n = 20) during the indicated time period. The time to develop MD in 50% of the inoculated animals (MD₅₀) is indicated (dashed line) and was significantly increased in the P6.1mut group (p = 0.0012).</p

Effect of the P6.1 mutation on telomerase activity.

<p>A) Schematic of the CR4–CR5 domain including a detailed representation of the P6.1 stem-loop. Structure of wild-type P6.1 (left) and mutant P6.1 stem-loop (P6.1 mut) (right) is shown. Nucleotide changes of the wt P6.1 stem-loop (blue) are shown in red. B) <i>In vitro</i> transcribed β-actin control RNA, wt vTR RNA, vTR containing the P6.1 mutation (P6.1) or a mutation in the template sequence (AU5) was analyzed on a 2% denaturing agarose-formaldehyde gel. Expected vTR size is indicated by the black arrow. C) Chicken TERT-His was translated <i>in vitro</i> using rabbit reticulocyte lysates and subsequently analyzed via western blotting using an anti-5x-His antibody. The expected size of TERT-His is indicated with the black arrow. D) Telomerase activity of the <i>in vitro</i> transcribed vTR variants was analyzed using gel based TRAP-assays. TRAP products and the internal control (IC) are indicated. The results shown are representative for three independent experiments showing similar results.</p

Model of chTR and vTR during MDV infection with vP6.1mut.

<p>chTR (gray) and wt vTR are able to interact with TERT (blue) and mediate telomerase activity, which is crucial for the survival of early MDV-transformed cells during initial crisis. P6.1mut vTR (Rose) is not able to interact with TERT and can, therefore, not contribute to telomerase activity. P6.1mut, as well as wt vTR, is able to interact with RPL22 (Red) and potentially also other factors (green) which mainly contributes to transformation and tumor dissemination.</p

Primers used for cloning and mutagenesis.

<p>Underlined sequences indicate restriction enzyme sites. Bold indicates mutated sequences.</p

Growth properties of viruses containing the P6.1 stem-loop mutation.

<p>A) Multi-step growth kinetics of wt vRB-1B, vP6.1mut and vP6.1rev were performed in triplicates and are shown as means with standard deviations (error bars). B) Plaque size assay. Results are shown for the three recombinant viruses as the relative mean plaque area in percent of 100 randomly selected plaques induced by each of the viruses with the corresponding standard deviations (error bars).</p

Infection with virulent virus induces TGF-beta⁺ Treg cells in vivo.

<p><b>(A)</b> A schematic diagram of animal trials is shown. Day old line P chicks were mock-infected or infected with the virulent virus via intra-tracheal route. Spleens were taken from 5 birds at different time points post infection. Percentages of <b>(B)</b> CD4⁺CD25⁺ T cells <b>(C)</b> CD4⁺CD25^high T cells <b>(D)</b> TGF-beta⁺CD4⁺ T cells <b>(E)</b> TGF-beta⁺CD4⁺CD25⁺ T cells are shown. <b>(F)</b> Absolute numbers of TGF-beta+ Treg cells are shown at 21 dpi. <b>(G)</b> Representative flow cytometry plots isolated from splenocytes of non-infected and MDV-infected chickens depicting the expression levels of TGF-beta within CD4⁺CD25^- or CD4⁺CD25⁺ T cell at 21 dpi. Dashed lines (Isotype control), solid line (CD4⁺CD25^- T cells), and shaded grey (CD4⁺CD25⁺ T cells). <b>(H)</b> Bar graph (mean ± SD) shows of MFIs of TGF-beta expression within CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells in non-infected and MDV infected chickens (n = 5). <b>(I)</b> The percentages of TGF-beta⁺CD4⁺ T cells within mononuclear cells isolated from the lungs at 4 dpi are depicted. * indicates a statistically significant difference (<i>P</i> < 0.05); ns, not significant.</p

Schematic of immunosuppression via induction of TGF-beta⁺ Treg during MDV infection.

<p>Upon infection with MDV via respiratory route, TGF-beta⁺ Treg cells, but not CD4⁺CD25⁺TGF-beta^negT cells, are expanded at 4 dpi in the lungs and at day 21 post infection in the spleens. There is an association between the induction of TGF-beta⁺ Treg cells and immunosuppression/pathogenicity of the disease. TGF-beta⁺ MDV-induced lymphoma cells express low levels of TGF-betaRI and II, while produce soluble inhibitory factors which can induce immunosuppression.</p