Prolonged colonisation with Escherichia coli O25:ST131 versus other extended-spectrum beta-lactamase-producing E. coli in a long-term care facility with high endemic level of rectal colonisation, the Netherlands, 2013 to 2014

The extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clone ST131 (ESBL-ST131) has spread in healthcare settings worldwide. The reasons for its successful spread are unknown, but might include more effective transmission and/or longer persistence. We evaluated the colonisation dynamics of ESBL-producing E. coli (ESBL-EC), including ESBL-ST131, in a long-term care facility (LTCF) with an unusually high prevalence of rectal ESBL-EC colonisation. During a 14-month period, rectal or faecal samples were obtained from 296 residents during six repetitive prevalence surveys, using ESBL-selective culture. Transmission rates, reproduction numbers, and durations of colonisation were compared for ESBL-ST131 vs other ESBL-EC. Furthermore, the likely time required for ESBL-ST131 to disappear from the LTCF was estimated. Over time, the endemic level of ESBL-ST131 remained elevated whereas other ESBL-EC returned to low-level prevalence, despite comparable transmission rates. Survival analysis showed a half-life of 13 months for ESBL-ST131 carriage, vs two to three months for other ESBL-EC (p < 0.001). Per-admission reproduction numbers were 0.66 for ESBL-ST131 vs 0.56 for other ESBL-EC, predicting a mean time of three to four years for ESBL-ST131 to disappear from the LTCF under current conditions. Transmission rates were comparable for ESBL-ST131 vs other ESBL-EC. Prolonged rectal carriage explained the persistence of ESBL-ST131 in the LTCF

Prolonged colonisation with Escherichia coli O25 : ST131 versus other extended-spectrum beta-lactamase-producing E. coli in a long-term care facility with high endemic level of rectal colonisation, the Netherlands, 2013 to 2014

The extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clone ST131 (ESBL-ST131) has spread in healthcare settings worldwide. The reasons for its successful spread are unknown, but might include more effective transmission and/or longer persistence. We evaluated the colonisation dynamics of ESBL-producing E. coli (ESBL-EC), including ESBL-ST131, in a long-term care facility (LTCF) with an unusually high prevalence of rectal ESBL-EC colonisation. During a 14-month period, rectal or faecal samples were obtained from 296 residents during six repetitive prevalence surveys, using ESBL-selective culture. Transmission rates, reproduction numbers, and durations of colonisation were compared for ESBL-ST131 vs other ESBL-EC. Furthermore, the likely time required for ESBL-ST131 to disappear from the LTCF was estimated. Over time, the endemic level of ESBL-ST131 remained elevated whereas other ESBL-EC returned to low-level prevalence, despite comparable transmission rates. Survival analysis showed a half-life of 13 months for ESBL-ST131 carriage, vs two to three months for other ESBL-EC (p < 0.001). Per-admission reproduction numbers were 0.66 for ESBL-ST131 vs 0.56 for other ESBL-EC, predicting a mean time of three to four years for ESBL-ST131 to disappear from the LTCF under current conditions. Transmission rates were comparable for ESBL-ST131 vs other ESBL-EC. Prolonged rectal carriage explained the persistence of ESBL-ST131 in the LTCF

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Off-tumor effect of inducible low affinity CD38-CAR T cells.

<p>MM patient bone marrow samples (n = 4) with ~20% MM cells were co-incubated with inducible low affinity (B1) CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g003" target="_blank">Fig 3A</a>. Depicted are the average CAR-dependent lysis of MM cells (CD138⁺/CD38⁺ ; open squares) and lysis of healthy non-MM cells (CD138^-/CD56^-/CD38^+/- ; grey diamonds) by inducible CD38-CAR T cells. Incubated with DOX for 24 hours 1000 ng/ml (upper left), 48 hours 1000 ng/ml (upper right), 24 hours 10 ng/ml (lower left), 48 hours 10 ng/ml (lower right). Presented is the pooled data from 4 independent experiments. Error bars indicate the mean +/- SEM. (Pt 2–5, same pts as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g004" target="_blank">Fig 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>).</p

Off-tumor effect of inducible CD38-CAR T cells.

<p>(A) Representative flow cytometry density plots of MM-BM with CD38⁺/CD138⁺ cells (MM) after treatment with inducible mock (+/- 1000 ng/ml DOX for 48 hours) and inducible high affinity (028) CD38-CAR T cells (- DOX or + 1000 ng/ml DOX for 48 hours and 0, 24 or 120 hours after DOX removal). (B) Pooled data obtained from the analysis of five MM patient bone marrow samples (patient 1–5, see for their phenotype data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>) with ~20% MM cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>) were co-incubated with inducible high affinity (028) CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g003" target="_blank">Fig 3A</a>. Shown are the mean CAR-dependent % lysis of MM (CD138⁺/CD38⁺ ; open squares) and % lysis of healthy non-MM cells (CD138^-/CD56^-/CD38^+/-; grey diamonds) by inducible CD38-CAR T cells. Incubated with DOX for 24 hours 1000 ng/ml (upper left), 48 hours 1000 ng/ml (upper right), 24 hours 10 ng/ml (lower left), 48 hours 10 ng/ml (lower right). Presented is the pooled data from 5 independent experiments. Error bars indicate the mean +/- SEM. (Pt 1–5, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>).</p

DOX induced CD38-CAR expression.

<p>(A) Schematic overview of constructs. The pRetroX-TRE3G vector with the P_TRE3GV inducible promoter controlling the transcription of Mock containing the marker LNGFR or the CD38-CAR (shown is high affinity scFv 028; the same vector design is also used for low affinity CARs A4 and B1), consisting of the single chain variable fragments, 4-1BB and CD3ζ and a LNGFR separated by a P2A sequence. These vectors were co-transduced with the pRetroX-TET-On 3G containing the transcription site for the transactivator protein rTta. (B) Representative flow cytometry density plots and histograms to determine CAR expression of the inducible CAR T cells, after 48 hours incubation with 0 or 1000 ng/ml DOX. The expression of the marker LNGFR was measured with an APC-conjugated antibody. CAR expression was measured by binding of his-tagged (HHHHHH) soluble CD38 (sCD38) protein to the ScFv domain, stained with PE-conjugated anti-His tag antibody.</p

DOX dose-dependent induction of CD38-CAR expression and anti-MM cytotoxicity.

<p>(A) Lysis of luciferase-transduced CD38⁺ MM cell lines UM9 and RPMI8226 after co-incubation with Mock and inducible, high affinity (028) CD38-CAR, after treatment with no or 1000 ng/ml DOX for 48 hours. Grey lines indicate the lysis by constitutively expressed high affinity (028) CD38-CAR T cells The BLI signal from surviving MM cells was measured after 16 hours using a luminometer and the percentage lysis was calculated as indicated in the material & methods. Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD (B) Mean fluorescent intensity (MFI) of the CAR measured by staining with soluble CD38-his after 48 hours incubation with 0, 1, 10, 100 or 1000 ng/ml DOX, Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD. (C) The cytotoxic activity of untreated or DOX treated inducible CD38-CAR T cells against luc+ MM cell line UM9 after 16 hours. Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD. In all panels * indicates p value <0.05 and ** <0.01 using one-way analysis of variance and subsequent multiple comparison.</p

Induction and decay kinetics of CD38-CAR expression.

<p>(A) schematic overview of CAR induction and decay assay. Black bars indicate the DOX incubation times, gray bars indicate the period of decay after the removal of DOX. (B and C) Representative results of five independent experiments of mean fluorescent intensity (MFI) of the CAR measured by staining with soluble his-tagged CD38 after 6, 24 or 48 hours incubation with (B) 10 or 1000 ng/ml DOX or 6, 24, 48 or 120 hours after washing of DOX (C) (an MFI of 600, observed by Mock cells was considered background expression). (D) A MM patient bone marrow sample with 20% MM cells was co-incubated with inducible, high affinity (028), CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule depicted in Fig 3A. are the CAR-dependent % lysis of CD138+/CD38+ MM cells (% lysed by CAR—% lysed by Mock). Presented is the representative data of n = 5. (E) Significant Pearson correlation of MFI of CAR expression as detected with soluble CD38 (sCD38) with % lysis of MM cells. High dose DOX R² = 0.60 and p = 0.012, low dose DOX R² = 0.61 and p = 0.015.</p

STAT3 labels a subpopulation of reactive astrocytes required for brain metastasis.

The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.We want to thank the CNIO Core Facilities for their excellent assistance. We also thank F.X. Real, O. Marin, M. Serrano, O. Fernandez-Capetillo and M. Soengas for critically reading the manuscript, P. Bos for advice with CD8+ T cell experiments, J. Massague (MSKCC) for the BrM cell lines, MEDA for Legasil, M. A. Perez (University of Copenhagen), H. Peinado (CNIO), M. Soengas (CNIO) and M. Squatrito (CNIO) for reagents. This work was supported by MINECO grants MINECO-Retos SAF201457243-R (M.V.), MINECO-Europa Excelencia SAF2015-62547-ERC (M.V.), FERO Grant for Research in Oncology (M.V.), Melanoma Research Alliance Young Investigator Award (M.V.), AECC Coordinated Translational Groups (M.V., E.M.-S. and S.R.y.C), SEOM (J.B.-B.), Pfizer WI190764 (J.B.-B.), Meda Pharma (J.B.-B.), Armangue Family Fund (JA.M. and J.B.-B.), La Caixa-Severo Ochoa International PhD Program Fellowship (L.Z.), FCT PhD Fellowship SFRH/BD/100089/2014 (C.M.), the Fulbright Program (W.B.). M.V. is a Ramon y Cajal Investigator (RYC-2013-13365). This work is dedicated to the memory of Maria Jesus Cortes Garin.S

PRPH2 mutation update: In silico assessment of 245 reported and 7 novel variants in patients with retinal disease

Mutations in PRPH2, encoding peripherin-2, are associated with the development of a wide variety of inherited retinal diseases (IRDs). To determine the causality of the many PRPH2 variants that have been discovered over the last decades, we surveyed all published PRPH2 variants up to July 2020, describing 720 index patients that in total carried 245 unique variants. In addition, we identified seven novel PRPH2 variants in eight additional index patients. The pathogenicity of all variants was determined using the ACMG guidelines. With this, 107 variants were classified as pathogenic, 92 as likely pathogenic, one as benign, and two as likely benign. The remaining 50 variants were classified as variants of uncertain significance. Interestingly, of the total 252 PRPH2 variants, more than half (n = 137) were missense variants. All variants were uploaded into the Leiden Open source Variation and ClinVar databases. Our study underscores the need for experimental assays for variants of unknown significance to improve pathogenicity classification, which would allow us to better understand genotype-phenotype correlations, and in the long-term, hopefully also support the development of therapeutic strategies for patients with PRPH2-associated IRD