Background To date, no immunological data on COVID-19 heterologous vaccination schedules in humans have been reported. We assessed the immunogenicity and reactogenicity of BNT162b2 (Comirnaty, BioNTech, Mainz, Germany) administered as second dose in participants primed with ChAdOx1-S (Vaxzevria, AstraZeneca, Oxford, UK). Methods We did a phase 2, open-label, randomised, controlled trial on adults aged 18–60 years, vaccinated with a single dose of ChAdOx1-S 8–12 weeks before screening, and no history of SARS-CoV-2 infection. Participants were randomly assigned (2:1) to receive either BNT162b2 (0·3 mL) via a single intramuscular injection (intervention group) or continue observation (control group). The primary outcome was 14-day immunogenicity, measured by immunoassays for SARS-CoV-2 trimeric spike protein and receptor binding domain (RBD). Antibody functionality was assessed using a pseudovirus neutralisation assay, and cellular immune response using an interferon-γ immunoassay. The safety outcome was 7-day reactogenicity, measured as solicited local and systemic adverse events. The primary analysis included all participants who received at least one dose of BNT162b2 and who had at least one efficacy evaluation after baseline. The safety analysis included all participants who received BNT162b2. This study is registered with EudraCT (2021-001978-37) and ClinicalTrials.gov ( NCT04860739 ), and is ongoing. Findings Between April 24 and 30, 2021, 676 individuals were enrolled and randomly assigned to either the intervention group (n=450) or control group (n=226) at five university hospitals in Spain (mean age 44 years [SD 9]; 382 [57%] women and 294 [43%] men). 663 (98%) participants (n=441 intervention, n=222 control) completed the study up to day 14. In the intervention group, geometric mean titres of RBD antibodies increased from 71·46 BAU/mL (95% CI 59·84–85·33) at baseline to 7756·68 BAU/mL (7371·53–8161·96) at day 14 (p<0·0001). IgG against trimeric spike protein increased from 98·40 BAU/mL (95% CI 85·69–112·99) to 3684·87 BAU/mL (3429·87–3958·83). The interventional:control ratio was 77·69 (95% CI 59·57–101·32) for RBD protein and 36·41 (29·31–45·23) for trimeric spike protein IgG. Reactions were mild (n=1210 [68%]) or moderate (n=530 [30%]), with injection site pain (n=395 [88%]), induration (n=159 [35%]), headache (n=199 [44%]), and myalgia (n=194 [43%]) the most commonly reported adverse events. No serious adverse events were reported. Interpretation BNT162b2 given as a second dose in individuals prime vaccinated with ChAdOx1-S induced a robust immune response, with an acceptable and manageable reactogenicity profile. Funding Instituto de Salud Carlos III. Translations For the French and Spanish translations of the abstract see Supplementary Materials section.

The role of viral load in the outcome of patients requiring hospital admission due to influenza is not well established. We aim to assess if there is an association between the viral load and the outcome in hospitalized patients with a confirmed influenza virus infection. A retrospective observational study including all adult patients who were hospitalized in our center with a confirmed influenza virus infection from January to May 2016. Viral load was measured by real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) cycle threshold (Ct) value on upper respiratory tract samples. Its value was categorized into three groups (low Ct,≤ 20; intermediate Ct,(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and high Ct, > 30). Two hundred thirty-nine patients were included. Influenza A/ H1N1pdm09 was isolated in 207 cases (86.6%). The mean Ct value was 26.69 ± 5.81. The viral load was higher in the unvaccinated group when compared with the vaccinated patients (Ct 25.17 ± 5.55 vs. 27.58 ± 4.97, p = 0.004). Only 27 patients (11.29%) presented a high viral load. Patients with a high viral load more often showed abnormal findings on chest X-ray (p = 0.015) and lymphopenia (p = 0.097). By contrast, there were no differences between the three groups (according to viral load), in associated pneumonia, respiratory failure, need for mechanical ventilation, sepsis, or in-hospital mortality. Our findings suggest that in patients admitted to the hospital with confirmed influenza virus infection (mostly A/H1N1pdm09), a high viral load is associated with a higher presence of abnormal findings on chest X-ray but not with a significant worse prognosis. In these cases, standardized quantitative PCR could be useful.

Objectives: There is increasing evidence that ferritin is a key marker of macrophage activation, but its potential role in influenza infection remains unexplored. Our aim was to assess whether hyperferritinaemia (ferritin 500 ng/mL) could be a marker of poor prognosis in hospitalized patients with confirmed influenza A infection. Methods: We prospectively recruited all hospitalized adult patients who tested positive for the influenza A rRT-PCR assay performed on respiratory samples in two consecutive influenza periods (2016e17 and 2017e18). Poor outcome was defined as the presence of at least one of the following: respiratory failure, admission to the intensive care unit, or in-hospital mortality. Results: Among 494 patients, 68 (14%) developed poor outcomes; 112 patients (23%) had hyperferritinaemia (39/68, 57% in the poor-outcome group versus 73/426, 17% in the remaining patients, p < 0.0001). Median serum ferritin levels were significantly higher in the subgroup of patients with poor outcomes (609 ng/mL, range 231e967 versus 217 ng/mL, range 140e394, p < 0.0001). In multivariate analysis, hyperferritinaemia was associated with a five-fold increase in the odds ratio of developing poor outcome. After adjusting for classic influenza risk factors, ferritin remained as a significant predictive factor in all exploratory models. Ferritin levels had a good discriminative capacity with an area under the ROC curve of 0.72 (95% confidence interval (CI) 0.65e0.8, p < 0.001) and an overall diagnostic accuracy for predicting poor outcome of 79.3% (95%CI 75.4e82.7%). Conclusions: Serum ferritin may discriminate a subgroup of patients with influenza infection who have a higher risk of developing a poor outcome.

Colorectal cancer (CRC) requires massive iron stores, but the complete mechanisms by which CRC modulates local iron handling and metabolically leverages iron are poorly understood. We demonstrate that the liver-derived, endocrine regulator of systemic iron balance, hepcidin, is activated ectopically in CRC. Hepcidin binds to the only known mammalian iron exporter ferroportin, resulting in degradation of ferroportin and intracellular iron trapping. Mice deficient for the hepcidin gene specifically in colon tumor epithelium exhibited significant decreases in tumor number, burden, and size compared to wild-type littermates in a sporadic model of CRC, whereas ferroportin deletion exacerbated these tumor parameters. To further understand the biochemical and metabolic utilization of iron in CRC, we subjected a three-dimensional patient-derived CRC tumor enteroid model to metabolomics and found that iron is prioritized in CRC for the production of nucleotides. These metabolomics findings were recapitulated in our hepcidin/ferroportin mouse CRC models. Mechanistically, our data suggest that a decrease in mitochondrial function alters nucleotide synthesis following iron chelation. Restoration of nucleotide metabolism with exogenous supplementation of nucleosides led to a partial rescue of growth in patient-derived tumor enteroids and CRC cell lines in the presence of an iron chelator. Moreover, aspartate, a critical metabolite which links mitochondrial respiration and nucleotide synthesis, also partially rescued growth of iron deficient CRC cells. Collectively, these data suggest that ectopic hepcidin in the tumor epithelium establishes an axis to degrade ferroportin and sequester iron in colorectal tumors in order to maintain the nucleotide pool and sustain proliferation.

Myc-associated zinc finger (MAZ) is a transcription factor highly upregulated in chronic inflammatory disease and several human cancers. In the present study, we found that MAZ protein is highly expressed in human ulcerative colitis and colon cancer. However, the precise role for MAZ in the progression of colitis and colon cancer is not well defined. To determine the function of MAZ, a novel mouse model of intestinal epithelial cell-specific MAZ overexpression was generated. Expression of MAZ in intestinal epithelial cells was sufficient to enhance inflammatory injury in two complementary models of colitis. Moreover, MAZ expression increased tumorigenesis in an model of inflammation-induced colon cancer and was important for growth of human colon cancer cell lines and Mechanistically, MAZ is critical in the regulation of oncogenic STAT3 signaling. MAZ-expressing mice have enhanced STAT3 activation in the acute response to colitis. Moreover, MAZ was essential for cytokine- and bacterium-induced STAT3 signaling in colon cancer cells. Furthermore, we show that STAT3 is essential for MAZ-induced colon tumorigenesis using a chemical inhibitor. These data indicate an important functional role for MAZ in the inflammatory progression of colon cancer through regulation of STAT3 signaling and suggest that MAZ is a potential therapeutic target to dampen STAT3 signaling in colon cancer.

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Hypoxia-inducible factor 2␣ (HIF2␣) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2␣ target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2␣ target genes in vivo. These observations indicate that different stimuli activate distinct subsets of HIF2␣ target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2␣'s transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2␣ or HIF1␣ target genes. Moreover, the highly homologous SMAD2 did not alter HIF2␣ transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2␣ activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.

Although hematological abnormalities have been described among patients with influenza virus infection, little is known about their impact on the outcome of the patients. The aim of this study was to assess the frequency and clinical impact of severe hematological abnormalities in patients with confirmed influenza virus infection. This was an observational retrospective study including all adult patients with diagnosis of influenza virus infection hospitalized from January to May 2016 in our institution. Influenza virus infection was diagnosed by means of rRT-PCR assay performed on respiratory samples. Poor outcome was defined as a composite endpoint in which at least one of the following criteria had to be fulfilled: (a) respiratory failure, (b) SOFA ≥2, or (c) death. Two hundred thirty-nine patients were included. Applying the HLH-04 criteria for the diagnosis of hemophagocytic syndrome, cytopenias (hemoglobin ≤9 g/dl, platelets <100,000/μl or neutrophils <1,000/μl) were present in 51 patients (21%). Patients with hematological abnormalities showed higher SOFA scores, respiratory failure, septic shock and in-hospital mortality than the remaining patients. The composite endpoint was present in 33.3% in the cytopenias group vs. 13.3% in the group without cytopenias (p=0.001). In a multivariate analysis, variables associated with the composite endpoint were: use of steroids prior to present admission (OR: 0.12; 95% CI: 0.015-0.96, p=0.046), presence of any hematological abnormality (OR: 3.54; 95% CI:1.66-7.51, p= 0.001), and LDH>225 U/l (OR:4.45; CI:1-19.71, p=0.049). Hematological abnormalities are not uncommon among hospitalized patients with influenza virus infection, and they are associated with a poorer outcome.