A randomized placebo-controlled trial of convalescent plasma for adults hospitalized with COVID-19 pneumonia

Passive immunotherapy with convalescent plasma may be the only available agent during the early phases of a pandemic. Here, we report safety and efficacy of high-titer convalescent plasma for COVID-19 pneumonia. Double-blinded randomized multicenter placebo-controlled trial of adult patients hospitalized with COVID-19 pneumonia. The intervention was COVID-19 convalescent plasma and placebo was saline allocated 2:1. The primary outcome was clinical status 14 days after the intervention evaluated on a clinical ordinal scale. The trial was registered at ClinicalTrials.Gov, NCT04345289, 14/04/2020. The CCAP-2 trial was terminated prematurely due to futility. Of 147 patients randomized, we included 144 patients in the modified intention-to-treat population. The ordinal clinical status 14 days post-intervention was comparable between treatment groups (odds ratio (OR) 1.41, 95% confidence interval (CI) 0.72–2.09). Results were consistent when evaluating clinical progression on an individual level 14 days after intervention (OR 1.09; 95% CI 0.46–1.73). No significant differences in length of hospital stay, admission to ICU, frequency of severe adverse events or all-cause mortality during follow-up were found between the intervention and the placebo group. Infusion of convalescent plasma did not influence clinical progression, survival or length of hospitalization in patients with COVID-19 pneumonia

HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

The mammalian Caudal-related homeobox transcription factor 2 (CDX2) plays a key role in the homeobox regulatory network and is essential in regulating the expression of several homeobox (HOX) genes during embryonic development, particularly in the gut. Genome-wide CDX2 chromatin immunoprecipitation analysis and expression data from Caco2 cells also suggests a role for CDX2 in the regulation of HOXB4 gene expression in the intestinal epithelium. Thus, the aim of this study was to investigate whether HOXB4 gene expression is regulated by CDX2 in the intestinal epithelium. We demonstrated binding of CDX2 to four different CDX2 binding sites in an enhancer region located upstream of the HOXB4 transcription start site. Mutations in the CDX2 binding sites reduced HOXB4 gene activity, and knock down of endogenous CDX2 expression by shRNA reduced HOXB4 gene expression. This is the first report demonstrating the CDX2 regulation of HOXB4 gene expression in the developed intestinal epithelium, indicating a possible role for HOXB4 in intestinal homeostasis

DNA oligo sequences used in this study.

<p>DNA oligo sequences used in this study.</p

Functional and EMSA analyses of CDX2 binding sites in the <i>HOXB4</i> enhancer region.

<p>Fig 3A) Positions of the four potential CDX2 binding sites and the EMSA probes covering these sites. Fig 3B) Reporter gene assays with four different luciferase constructs containing the <i>HOXB4</i> enhancer with wild-type CDX2 binding sites or four distinct reporter constructs containing individual mutations in the four CDX2 binding sites (<i>HOXB4</i> site-1, -2, -3 or 4). The mean (n = 4) and SD are shown. *<i>P</i><0.05 and **<i>P</i><0.01. Fig 3C–3F) EMSAs with probes for the four different CDX2 binding sites (HOXB4 site-1, -2, -3 and 4). One hundred-fold unlabeled probe was added to the competition reactions as well as sucrase isomaltase (SI) or unspecific probe (unspec). Influenza hemagglutinin (HA) antibody (negative control) or CDX2 antibody (CDX2) was used in the supershift assay. Non-specific bands and supershifts indicated by black arrows.</p

Illustration of the CDX2 binding region in the <i>HOXB4</i> enhancer region and analysis of CDX2 interactions with the <i>HOXB4</i> enhancer region.

<p>Fig 1A) Genome browser CDX2 ChIP-seq peaks from a dataset generated by Boyd et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164555#pone.0164555.ref017" target="_blank">17</a>]. Fig 1B) ChIP-PCR with primers covering the CDX2 binding region within the <i>HOXB4</i> enhancer. Equal amounts of input DNA were used for CDX2 ChIP-PCR. Hemagglutinin (HA) was used as a negative control. The mean (n = 4) and SD are shown. *<i>P</i>< 0.05.</p

The effects of <i>CDX2</i> knock down and overexpression on HOXB4 expression in the SW480 cell line and functional analysis of 4 different CDX2 binding sites in the <i>HOXB4</i> enhancer region.

<p>Fig 2A) Western blot analysis of CDX2 expression levels in SW480 cells treated with scrambled shRNA and cells in which <i>CDX2</i> was knocked down. GADPH was used as a control to analyze equal amounts of total cellular protein. Fig 2B) The effects of <i>CDX2</i> knock down on the promoter activities of reporter constructs containing the <i>HOXB4</i> promoter alone or the <i>HOXB4</i> promoter including the enhancer region. Fig 2C) Transient transfection of the <i>HOXB4</i> promoter and enhancer reporter construct in SW480 cells with and without co-transfection of a CDX2 expression plasmid. The mean (n = 4) and SD are shown. *<i>P</i><0.05 and **P<0.01</p