COVID-19 Severity and Survival over Time in Patients with Hematologic Malignancies: A Population-Based Registry Study

Mortality rates for COVID-19 have declined over time in the general population, but data in patients with hematologic malignancies are contradictory. We identified independent prognostic factors for COVID-19 severity and survival in unvaccinated patients with hematologic malignancies, compared mortality rates over time and versus non-cancer inpatients, and investigated post COVID-19 condition. Data were analyzed from 1166 consecutive, eligible patients with hematologic malignancies from the population-based HEMATO-MADRID registry, Spain, with COVID-19 prior to vaccination roll-out, stratified into early (February–June 2020; n = 769 (66%)) and later (July 2020–February 2021; n = 397 (34%)) cohorts. Propensity-score matched non-cancer patients were identified from the SEMI-COVID registry. A lower proportion of patients were hospitalized in the later waves (54.2%) compared to the earlier (88.6%), OR 0.15, 95%CI 0.11–0.20. The proportion of hospitalized patients admitted to the ICU was higher in the later cohort (103/215, 47.9%) compared with the early cohort (170/681, 25.0%, 2.77; 2.01–3.82). The reduced 30-day mortality between early and later cohorts of non-cancer inpatients (29.6% vs. 12.6%, OR 0.34; 0.22–0.53) was not paralleled in inpatients with hematologic malignancies (32.3% vs. 34.8%, OR 1.12; 0.81–1.5). Among evaluable patients, 27.3% had post COVID-19 condition. These findings will help inform evidence-based preventive and therapeutic strategies for patients with hematologic malignancies and COVID-19 diagnosis.Depto. de MedicinaFac. de MedicinaTRUEFundación Madrileña de Hematología y HemoterapiaFundación Leucemia y LinfomaAsociación Madrileña de Hematología y Hemoterapiapu

Translocated LPS Might Cause Endotoxin Tolerance in Circulating Monocytes of Cystic Fibrosis Patients

Cystic Fibrosis (CF) is an inherited pleiotropic disease that results from abnormalities in the gene codes of a chloride channel. The lungs of CF patients are chronically infected by several pathogens but bacteraemia have rarely been reported in this pathology. Besides that, circulating monocytes in CF patients exhibit a patent Endotoxin Tolerance (ET) state since they show a significant reduction of the inflammatory response to bacterial stimulus. Despite a previous description of this phenomenon, the direct cause of ET in CF patients remains unknown. In this study we have researched the possible role of microbial/endotoxin translocation from a localized infection to the bloodstream as a potential cause of ET induction in CF patients. Plasma analysis of fourteen CF patients revealed high levels of LPS compared to healthy volunteers and patients who suffer from Chronic Obstructive Pulmonary Disease. Experiments in vitro showed that endotoxin concentrations found in plasma of CF patients were enough to induce an ET phenotype in monocytes from healthy controls. In agreement with clinical data, we failed to detect bacterial DNA in CF plasma. Our results suggest that soluble endotoxin present in bloodstream of CF patients causes endotoxin tolerance in their circulating monocytes

CTXφ: Exploring new alternatives in host factor-mediated filamentous phage replications

International audienceFor a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXφ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXφ and VGJφ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages

CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase.

International audienceThe Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU

Impaired replication of CTXϕ in ∆<i>hupB</i> cells.

<p><b>(A)</b> Q-PCR analysis of the number ssDNA and dsDNA copies of RS2 in the indicated strain<i>s</i>. The analysis was performed on the total DNA of cells that were grown under selection pressure at 30°C to an OD_600nm of 0.3. Data represent the mean of two independent experiments. <b>(B)</b> Phage maintenance was measured in the indicated strain<i>s</i> after 5 h of growth in LB without selection. Serial dilutions of 10 were dropped on plates with or without selection pressure as indicated. <b>(C)</b> Relative efficient of RS2 integration in <i>lac</i>Z::<i>dif</i>1, <i>lac</i>Z::<i>dif</i>1 ∆<i>hupA</i> and <i>lac</i>Z::<i>dif</i>1 ∆<i>hupB</i> cells. Integration was monitored after overnight growth in LB at 37°C. Data represents the mean and standard deviation of 3 independent experiments. A t-Test was used to determine the probability, <i>p</i>, that the samples came from similar distributions (*, <i>p</i><0.1; ***, <i>p</i><0.001). <b>(D)</b> Relative efficiency of integration of a non-replicative vector harbouring the <i>attP</i> of CTXϕ delivered by conjugation in <i>lac</i>Z::<i>dif</i>1, <i>lac</i>Z::<i>dif</i>1 ∆<i>hupA</i>, <i>lac</i>Z::<i>dif</i>1 ∆<i>hupB</i>, <i>lac</i>Z::<i>dif</i>1 ∆<i>hupAB</i> cells. Integration was monitored directly after conjugation. Data represents the mean and standard deviation of 3 independent experiments. <b>(E)</b> Relative production of CTXϕ particles by ∆<i>hupB</i> ∆<i>xerC</i> cells. ∆<i>xerC</i> recipient cells were incubated for 20’ in the filtered supernatant ∆<i>xerC</i> and ∆<i>hupB</i> ∆<i>xerC</i> donor cells harbouring pCTX-Kn. Data represents the mean and standard deviation of 3 independent experiments. The detection limit of the experiment is indicated by a dotted line.</p

Replication of CTXϕ depends on UvrD.

<p><b>(A)</b> Relative colony-forming ability after conjugation with RS2 in the indicated strain<i>s</i>. After 3 h of conjugation serial dilutions were plated on chloramphenicol and incubated overnight at 37°C. Relative colony forming units (cfu) correspond to the ratio of the number of colonies obtained in the indicated strain over the mean number of colonies obtained in ∆<i>xerC</i> cells. Results are shown in a logarithmic scale and represent the mean and standard deviation of 3 independent experiments. The detection limit of the experiment is indicated by a dotted line. <b>(B)</b> Complementation assay of Δ<i>xerC</i> Δ<i>uvrD</i> cells with a pBAD vector carrying the <i>V</i>. <i>cholerae uvrD</i> gene (pBAD-<i>uvrD</i>). RS2 was conjugated into Δ<i>xerC</i> and Δ<i>xerC</i> Δ<i>uvrD</i> cells harbouring pBAD or pBAD-<i>uvrD</i> and plated on LB supplemented with ampicillin, chloramphenicol and 0.2% of arabinose. Plates were incubated overnight at 37°C. <b>(C)</b> Relative colony-forming ability of <i>lac</i>Z::<i>dif</i>1, <i>lac</i>Z::<i>dif</i>1 Δ<i>rep</i> and <i>lac</i>Z::<i>dif</i>1 Δ<i>uvrD</i> cells after 3 h of conjugation with RS2. Serial dilutions were plated on chloramphenicol and incubated overnight at 37°C. Relative colony forming units (cfu) correspond to the ratio of the number of colonies obtained in the indicated strain over the mean number of colonies obtained in Δ<i>xerC</i> cells. Results are shown in a logarithmic scale and represent the mean and standard deviation of 3 independent experiments. The detection limit of the experiment is indicated by a dotted line. <b>(D)</b> Phenotype of colonies obtained after RS2 integration in <i>lac</i>Z::<i>dif</i>1 (Top) and <i>lac</i>Z::<i>dif</i>1 Δ<i>uvrD</i> (Bottom) cells.</p

Cleavage of CTXϕ <i>ori(+)</i> by RstA depends on HU.

<p><b>(A)</b> Scheme depicting the primer extension assay used to monitor RstA cleavage. Red arrows in opposite direction depict potential loops in the <i>ori(+)</i> region. The <i>rstR</i> gene, the NotI site and the location of the primer used in the primer extension are shown. <b>(B)</b> RstA activity in the indicated strains. Top left. Electrophoresis of the products was performed with a 6% polyacrylamide/8M urea gel. Lane 1–4: dideoxy sequence ladder. Top right: schematic representation of <i>ori(+)</i> loop 2. Bottom: relative intensity of the primer extension profiles. Black triangle: position of the nick. <b>(C)</b> Schematic representing the formation of a dsDNA break when replication forks originating from <i>ori</i>^<i>101</i> encounter a nick created by RstA. Black and red stars depict Rep^<i>TS</i> and RstA proteins, respectively. <b>(D)</b> Relative colony-forming ability after conjugation. Column 1–3: pSC101-RS2 was conjugated in the indicated strains to Δ<i>xerC</i>, Δ<i>xerC</i> Δ<i>hupAB</i>, Δ<i>xerC</i> Δ<i>uvrD</i>, <i>and</i> Δ<i>xerC</i> Δ<i>hupAB</i> Δ<i>uvrD</i> cells, respectively; Column 5: pSC101-RS2 Δ<i>rstA</i> was conjugated in Δ<i>xerC</i> cells. After 3 h of conjugation, serial dilutions were plated on spectinomycin and incubated overnight at 30°C. Relative colony forming units (cfu) correspond to the ratio of the number of colonies obtained in the indicated strain over the mean number of colonies obtained in Δ<i>xerC</i> cells. Results are shown in a logarithmic scale and represent the mean and standard deviation of 3 independent experiments. The detection limit of the experiment is indicated by a dotted line. <b>(E)</b> Q-PCR analysis of the number of pSC101-RS2 ssDNA and dsDNA copies in Δ<i>xerC</i>, Δ<i>xerC</i> Δ<i>hupAB</i> and Δ<i>xerC</i> Δ<i>hupAB</i> Δ<i>uvrD</i> cells, and of Δ<i>rstA</i> pSC101-RS2 ssDNA and dsDNA in Δ<i>xerC</i> cells. The analysis was performed on the total DNA of cells that were grown under selective pressure at 30°C to an OD_600nm of 0.3. Data represent the mean of two independent experiments.</p

HU is essential for CTXϕ replication.

<p><b>(A and B)</b> Relative colony-forming ability after conjugation with a replicative form of RS2 in the indicated strains. After 3 h of conjugation, serial dilutions were plated on chloramphenicol and incubated overnight at 42°C (A) and 37°C (B). Relative colony forming units (cfu) correspond to the ratio of the number of colonies obtained in the indicated strain over the mean number of colonies obtained in ∆<i>xerC</i> cells. Results are shown in a logarithmic scale and represent the mean and standard deviation of 3 independent experiments. The detection limit of the experiment is indicated by a dotted line. <b>(C and D)</b> Colonies obtained at 37°C were re-streaked on plate and incubated overnight at 42°C (C) and 37°C (D). <b>(E)</b> Complementation assay of Δ<i>hupAB</i> cells with a pUC18 vector carrying the <i>V</i>. <i>cholerae hupA</i> gene (pUC-<i>hupA</i>) <i>or hupB</i> gene (pUC-<i>hupB</i>). RS2 was conjugated into Δ<i>xerC</i> Δ<i>hupAB</i> + pUC18, Δ<i>xerC</i> Δ<i>hupAB</i> + pUC-<i>hupA</i> and Δ<i>xerC</i> Δ<i>hupAB</i> + pUC-<i>hupB</i>. The conjugants were then streaked on a plate supplemented with ampicillin and chloramphenicol. The plate was incubated overnight at 37°C.</p

Rolling-Circle Replication is central to the life cycle of CTXϕ.

<p>Schematic diagram showing key steps in the life cycle of CTXϕ. CTXϕ infection requires the host-encoded toxin co-regulated pilus (TCP) and TolQRA proteins (1). After its release in the cytoplasm of its host, CTXϕ ssDNA is converted into a dsDNA by the host machineries (2). Rolling circle replication (RCR) of the phage depends on a single phage-encoded protein, RstA, and on the host machinery (3). New CTXϕ particle secretion depends on the host outer membrane protein EpsD (4). RstA production is under the control of the SOS response (5). Integration of CTXϕ depends on the host Xer machinery (6) and the accessory protein EndoIII (7). A process akin to RCR permits the production of free copies of CTXϕ ssDNA when the phage genome is integrated in tandem (8).</p

Screen for host factors implicated in CTXϕ replication.

<p><b>(A)</b> Scheme of the conjugation of the pSC101-RS2 hybrid into a <i>lac</i>Z::<i>dif</i>1 reporter strain. <b>(B)</b> Schematic representation of the colonies obtained in different genetic backgrounds. <i>f</i>: relative frequency of formation of each type of depicted colony; (<i>i)</i>: colony formed upon direct integration; (<i>ii)</i>: colony obtained after RCR amplification of the phage DNA; (<i>iii)</i>: colony obtained when host factors implicated in integration are disrupted; (iv): colony obtained when host factors implicated in RCR are disrupted. <b>(C)</b> Scheme of the VC1919 region. Open triangles indicate the position of insertion of the transposon that impeded pSC101-RS2 RCR.</p