Role of T198 Modification in the Regulation of p27Kip1 Protein Stability and Function

The tumor suppressor gene p27Kip1 plays a fundamental role in human cancer progression. Its expression and/or functions are altered in almost all the different tumor histotype analyzed so far. Recently, it has been demonstrated that the tumor suppression function of p27 resides not only in the ability to inhibit Cyclins/CDKs complexes through its N-terminal domain but also in the capacity to modulate cell motility through its C-terminal portion. Particular interest has been raised by the last amino-acid, (Threonine 198) in the regulation of both protein stability and cell motility

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.</p

Liver Gene Transfer of Interkeukin-15 Constructs That Become Part of Circulating High Density Lipoproteins for Immunotherapy

<div><p>Apolipoprotein A-I (Apo A-I) is a major component of high density lipoproteins (HDL) that transport cholesterol in circulation. We have constructed an expression plasmid encoding a chimeric molecule encompassing interleukin-15 (IL-15) and Apo A-I (pApo-hIL15) that was tested by hydrodynamic injections into mice and was co-administered with a plasmid encoding the sushi domain of IL-15Rα (pSushi) in order to enhance IL-15 trans-presentation and thereby bioactivity. The pharmacokinetics of the Apo A-I chimeric protein were much longer than non-stabilized IL-15 and its bioactivity was enhanced in combination with IL-15Rα Sushi. Importantly, the APO-IL-15 fusion protein was incorporated in part into circulating HDL. Liver gene transfer of these constructs increased NK and memory-phenotype CD8 lymphocyte numbers in peripheral blood, spleen and liver as a result of proliferation documented by CFSE dilution and BrdU incorporation. Moreover, the gene transfer procedure partly rescued the NK and memory T-cell deficiency observed in IL-15Rα^−/− mice. pApo-hIL15+ pSushi gene transfer to the liver showed a modest therapeutic activity against subcutaneously transplanted MC38 colon carcinoma tumors, that was more evident when tumors were set up as liver metastases. The improved pharmacokinetic profile and the strong biological activity of APO-IL-15 fusion protein holds promise for further development in combination with other immunotherapies.</p> </div

Liver gene transfer of APO-IL-15 and IL-15Rα sushi domain results in extended half-life and enhanced bioactivity.

<p>(A) Mice (n = 3 per group) were hydrodynamically injected with the indicated expression plasmids and serum concentrations of human IL-15 were sequentially monitored by ELISA. Mean ± SEM from one representative experiment out of three similarly performed. (B) IL-15 and Apo A-I mRNA expression quantified by real-time RT-PCR referred to β actin at the given time-points following hydrodynamic injection of the indicated plasmids as in A. (C) Plasma samples of mice hydrodynamically treated with the indicated plasmids were fractioned by centrifugation into the different lipoprotein particles and lipid-depleted plasma. IL-15 content in each fraction was monitored by ELISA. Results are from two pooled experiments performed with 4 and 6 mice per group. The inset represents the Apo A-I concentration in the indicated fractions. (D) IL-15 bioactivity measured on CTLL2 cells by ³H-Thy incorporation when testing the indicated dilutions of the serum and the HDL concentrated fractions from equivalent volumes of serum. Concentration were adjusted to nmol/mL using IL-15 concentration as assessed by ELISA. (E) CTLL-2 proliferation in response to sequentially diluted plasma samples in which IL-15 content had been measured by ELISA as in D. When indicated, mice had been hydrodynamically given pApo-hIL15 with or without combination with a plasmid encoding the IL-15Rα sushi domain. Data represent mean±SEM of four-replicated samples. The experiment was repeated at least three times with comparable results. Commercial human rIL-15 is included as a positive control and the discontinuous line represents thymidine incorporation to CTLL2 exposed to the serum of a mouse treated with pApo as a negative control.</p

Antitumor effects following the transfer of IL-15 constructions against MC38 tumors metastatic to the liver.

<p>Mice injected intraesplenically with 5x10⁵ MC38 cells upon laparotomy were treated by hydrodynamic injections of the indicated plasmids one day later. On day 19 mice were euthanized and (A) liver surface was inspected to count macroscopic metastases observed in the surface of the liver under magnifying lens and data are represented on individual basis (*** indicates p<0.0001 between pApo-hIL15+ pSushi and the rest of the groups in Bonferroni test following one-way ANOVA test). (B) The weights of the spleens that also grafted tumors were recorded. Representative images of spleen tumors in the indicated groups are provided. The experiment shown is representative of two similarly performed (* indicates p<0.05 between pApo-hIL15+ pSushi and pApo groups in a t-student test).</p

Immunotherapeutic activity of liver gene transfer with the APO-IL-15 constructions for established subcutaneous MC38 tumors.

<p>MC38 bearing mice were treated on day six after tumor cell inoculation with 10 µg of the indicated plasmids by hydrodynamic injection. (A) Individual follow-up of tumor sizes upon treatment with each plasmid. Graphics represent pooled data from 3 independent experiments. Fraction of surviving mice is indicated for each experimental group. Differences in tumor growth between the pApo-hIL15+ pSushi and the pApo group were found (p<0.001) in a nonlinear mixed effect model calculated with Monolix software. (B) Survival curves of the experimental groups from panel A (* indicates p<0.05 between pApo-hIL15+ pSushi and pApo in a Gehan-Breslow-Wilcoxon Survival Test).</p

APO-IL-15 and IL-15Rα sushi domain gene co-transfer induces proliferation of adoptively transferred CD8 and NK lymphocytes.

<p>Mice were gene-transferred by hydrodynamic injections of the indicated plasmids which were 24 h later intravenously injected with CD8⁺ T cells or NK1.1⁺ NK cells preloaded with CFSE. Proliferation was monitored by CFSE dilution of the indicated electronically gated lymphocyte subsets (dot plots A and C). Results pooled from two independent experiments included two mice per experimental group (B and D). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052370#pone.0052370.s007" target="_blank">Figure S7</a> shows proliferative effects on endogenous lymphocytes studied by BrdU intake.</p

Figure 3. Gene transfer of APO-IL-15 and IL-15Rα sushi domain increase memory-phenotype CD8⁺ T cells and NK cells.

<p>(A) Absolute numbers of the indicated lymphocyte subsets in the spleen of mice treated with the indicated plasmids 5 days before splenectomy. Each point shows an individual mouse. This experiment was repeated twice with comparable results. (*** indicates p<0.0001 in a one-way ANOVA test followed by Bonferroni corrections). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052370#pone.0052370.s005" target="_blank">Figure S5</a> shows a time course analysis of these parameters (B) Experiments as in A measuring numbers of intrahepatic mononuclear leukocytes with the indicated surface phenotypes. Each point represents an individual mouse (*** indicates p<0.0001 in a one-way ANOVA test followed by Bonferroni corrections).</p

Partial rescue of the phenotype of IL-15Rα^−/− mice in NK cells and memory-phenotype CD8 cells.

<p>WT C57Bl/6 mice (WT) or IL-15Rα^−/− syngenic mice (IL-15Rα^−/−) were injected with the indicated plasmids by hydrodynamic injection. Four days later splenocytes were immunostained. (A) CD3⁺ CD8⁺ dot plots with the percentage of events in the gated rectangles. Results reflect the percentage referred to total spenocytes (B) Histograms showing CD44 specific immunofluorescence on the cells gated in A to assess the percentage of CD44^high CD8⁺ memory-phenotype T cells. (C) CD3 NK1.1 double staining showing the percentage of events in each dot plot quadrant. Histograms and dot plots were taken from a representative individual mouse. (D) Grouped sets of data from other individuals. Confirmatory results in a series of mice in B6129 background are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052370#pone.0052370.s008" target="_blank">figure S8</a>.</p