Transcriptomic responses of Mediterranean sponges upon encounter with symbiont microbial consortia.

BACKGROUND: Sponges (phylum Porifera) constantly interact with microbes. They graze on microbes from the water column by filter-feeding and they harbor symbiotic partners within their bodies. In experimental setups, sponges take up symbionts at lower rates compared with seawater microbes. This suggests that sponges have the capacity to differentiate between microbes and preferentially graze in non-symbiotic microbes, although the underlying mechanisms of discrimination are still poorly understood. Genomic studies showed that, compared to other animal groups, sponges present an extended repertoire of immune receptors, in particular NLRs, SRCRs, and GPCRs, and a handful of experiments showed that sponges regulate the expression of these receptors upon encounter with microbial elicitors. We hypothesize that sponges may rely on differential expression of their diverse repertoire of poriferan immune receptors to sense different microbial consortia while filter-feeding. To test this, we characterized the transcriptomic response of two sponge species, Aplysina aerophoba and Dysidea avara, upon incubation with microbial consortia extracted from A. aerophoba in comparison with incubation with seawater microbes. The sponges were sampled after 1 h, 3 h, and 5 h for RNA-Seq differential gene expression analysis. RESULTS: D. avara incubated with A. aerophoba-symbionts regulated the expression of genes related to immunity, ubiquitination, and signaling. Within the set of differentially-expressed immune genes we identified different families of Nucleotide Oligomerization Domain (NOD)-Like Receptors (NLRs). These results represent the first experimental evidence that different types of NLRs are involved in microbial discrimination in a sponge. In contrast, the transcriptomic response of A. aerophoba to its own symbionts involved comparatively fewer genes and lacked genes encoding for immune receptors. CONCLUSION: Our work suggests that: (i) the transcriptomic response of sponges upon microbial exposure may imply "fine-tuning" of baseline gene expression as a result of their interaction with microbes, (ii) the differential response of sponges to microbial encounters varied between the species, probably due to species-specific characteristics or related to host's traits, and (iii) immune receptors belonging to different families of NLR-like genes played a role in the differential response to microbes, whether symbionts or food bacteria. The regulation of these receptors in sponges provides further evidence of the potential role of NLRs in invertebrate host-microbe interactions. The study of sponge responses to microbes exemplifies how investigating different animal groups broadens our knowledge of the evolution of immune specificity and symbiosis.

Immune cell kinetics and antibody response in COVID-19 patients with low-count monoclonal B-cell lymphocytosis.

Low-count monoclonal B-cell lymphocytosis (MBLlo ) has been associated with an underlying immunodeficiency and has recently emerged as a new risk factor for severe COVID-19. Here, we investigated the kinetics of immune cell and antibody responses in blood during COVID-19 of MBLlo versus non-MBL patients. For this study, we analyzed the kinetics of immune cells in blood of 336 COVID-19 patients (74 MBLlo and 262 non-MBL), who had not been vaccinated against SARS-CoV-2, over a period of 43 weeks since the onset of infection, using high-sensitivity flow cytometry. Plasma levels of anti-SARS-CoV-2 antibodies were measured in parallel by ELISA. Overall, early after the onset of symptoms, MBLlo COVID-19 patients showed increased neutrophil, monocyte, and particularly, plasma cell (PC) counts, whereas eosinophil, dendritic cell, basophil, and lymphocyte counts were markedly decreased in blood of a variable percentage of samples, and with a tendency toward normal levels from week +5 of infection onward. Compared with non-MBL patients, MBLlo COVID-19 patients presented higher neutrophil counts, together with decreased pre-GC B-cell, dendritic cell, and innate-like T-cell counts. Higher PC levels, together with a delayed PC peak and greater plasma levels of anti-SARS-CoV-2-specific antibodies (at week +2 to week +4) were also observed in MBLlo patients. In summary, MBLlo COVID-19 patients share immune profiles previously described for patients with severe SARS-CoV-2 infection, associated with a delayed but more pronounced PC and antibody humoral response once compared with non-MBL patients.

SARS-CoV-2 Seroprevalence and Antibody Kinetics Among Health Care Workers in a Spanish Hospital After 3 Months of Follow-up.

BACKGROUND: At the COVID-19 spring 2020 pandemic peak in Spain, prevalence of SARS-CoV-2 infection in a cohort of 578 randomly selected health care workers (HCWs) from Hospital Clínic de Barcelona was 11.2%. METHODS: A follow-up survey 1 month later (April-May 2020) measured infection by rRT-PCR and IgM, IgA, and IgG to the receptor-binding domain of the spike protein by Luminex. Antibody kinetics, including IgG subclasses, was assessed until month 3. RESULTS: At month 1, the prevalence of infection measured by rRT-PCR and serology was 14.9% (84/565) and seroprevalence 14.5% (82/565). We found 25 (5%) new infections in 501 participants without previous evidence of infection. IgM, IgG, and IgA levels declined in 3 months (antibody decay rates 0.15 [95% CI, .11-.19], 0.66 [95% CI, .54-.82], and 0.12 [95% CI, .09-.16], respectively), and 68.33% of HCWs had seroreverted for IgM, 3.08% for IgG, and 24.29% for IgA. The most frequent subclass responses were IgG1 (highest levels) and IgG2, followed by IgG3, and only IgA1 but no IgA2 was detected. CONCLUSIONS: Continuous and improved surveillance of SARS-CoV-2 infections in HCWs remains critical, particularly in high-risk groups. The observed fast decay of IgA and IgM levels has implications for seroprevalence studies using these isotypes.

A Two-Photon Probe Based on Naphthalimide-Styrene Fluorophore for the In Vivo Tracking of Cellular Senescence.

Cellular senescence is a state of stable cell cycle arrest that can negatively affect the regenerative capacities of tissues and can contribute to inflammation and the progression of various aging-related diseases. Advances in the in vivo detection of cellular senescence are still crucial to monitor the action of senolytic drugs and to assess the early onset or accumulation of senescent cells. Here, we describe a naphthalimide-styrene-based probe (HeckGal) for the detection of cellular senescence both in vitro and in vivo. HeckGal is hydrolyzed by the increased lysosomal ß-galactosidase activity of senescent cells, resulting in fluorescence emission. The probe was validated in vitro using normal human fibroblasts and various cancer cell lines undergoing senescence induced by different stress stimuli. Remarkably, HeckGal was also validated in vivo in an orthotopic breast cancer mouse model treated with senescence-inducing chemotherapy and in a renal fibrosis mouse model. In all cases, HeckGal allowed the unambiguous detection of senescence in vitro as well as in tissues and tumors in vivo. This work is expected to provide a potential technology for senescence detection in aged or damaged tissues.

Pre- and post-operative anti-PD-L1 plus anti-angiogenic therapies in mouse breast or renal cancer models of micro- or macro-metastatic disease.

BACKGROUND: There are phase 3 clinical trials underway evaluating anti-PD-L1 antibodies as adjuvant (postoperative) monotherapies for resectable renal cell carcinoma (RCC) and triple-negative breast cancer (TNBC); in combination with antiangiogenic VEGF/VEGFR2 inhibitors (e.g., bevacizumab and sunitinib) for metastatic RCC; and in combination with chemotherapeutics as neoadjuvant (preoperative) therapies for resectable TNBC. METHODS: This study investigated these and similar clinically relevant drug combinations in highly translational preclinical models of micro- and macro-metastatic disease that spontaneously develop after surgical resection of primary kidney or breast tumours derived from orthotopic implantation of murine cancer cell lines (RENCAluc or EMT-6/CDDP, respectively). RESULTS: In the RENCAluc model, adjuvant sunitinib plus anti-PD-L1 improved overall survival compared to either drug alone, while the same combination was ineffective as early therapy for unresected primary tumours or late-stage therapy for advanced metastatic disease. In the EMT-6/CDDP model, anti-PD-L1 was highly effective as an adjuvant monotherapy, while its combination with paclitaxel chemotherapy (with or without anti-VEGF) was most effective as a neoadjuvant therapy. CONCLUSIONS: Our preclinical data suggest that anti-PD-L1 plus sunitinib may warrant further investigation as an adjuvant therapy for RCC, while anti-PD-L1 may be improved by combining with chemotherapy in the neoadjuvant but not the adjuvant setting of treating breast cancer.

Anti-oncostatin M antibody inhibits the pro-malignant effects of oncostatin M receptor overexpression in squamous cell carcinoma.

The oncostatin M (OSM) receptor (OSMR) shows frequent gene copy number gains and overexpression in cervical squamous cell carcinomas (SCCs), associated with adverse clinical outcomes. In SCC cells that overexpress OSMR, the major ligand OSM induces multiple pro-malignant effects, including invasion, secretion of angiogenic factors, and metastasis. Here, we demonstrate, for the first time, that OSMR overexpression in SCC cells activates cell-autonomous feed-forward signalling, via further expression of OSMR and OSM and sustained STAT3 activation, despite expression of the negative regulator suppressor of cytokine signalling 3 (SOCS3). The pro-malignant effects associated with OSMR overexpression are critically mediated by JAK-STAT3 activation, which is induced by exogenous OSM and also by autocrine OSM-OSMR interactions. Importantly, specific inhibition of OSM-OSMR interactions by neutralizing antibodies significantly inhibits STAT3 activation and feed-forward signalling, leading to reduced invasion, angiogenesis, and metastasis. Our findings are supported by data from 1254 clinical SCC samples, in which OSMR levels correlated with multiple cognate genes, including OSM, STAT3, and downstream targets. These data strongly support the development of OSM-OSMR-blocking antibodies as biologically targeted therapies against SCCs of the cervix and other anatomical sites. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Host-targeted RAD-Seq reveals genetic changes in the coral Oculina patagonica associated with range expansion along the Spanish Mediterranean coast.

Many organisms are expanding their ranges in response to changing environmental conditions. Understanding the patterns of genetic diversity and adaptation along an expansion front is crucial to assessing a species' long-term success. While next-generation sequencing techniques can reveal these changes in fine detail, ascribing them to a particular species can be difficult for organisms that live in close association with symbionts. Using a novel modified restriction site-associated DNA sequencing (RAD-Seq) protocol to target coral DNA, we collected 595 coral-specific single nucleotide polymorphisms from 189 colonies of the invasive coral Oculina patagonica from the Spanish Mediterranean coast, including established core populations and two expansion fronts. Surprisingly, populations from the recent northern expansion are genetically distinct from the westward expansion and core populations and also harbour greater genetic diversity. We found that temperature may have driven adaptation along the northern expansion, as genome scans for selection found three candidate loci associated with temperature in the north but none in the west. We found no genomic signature of selection associated with artificial substrate, which has been proposed for explaining the rapid spread of O. patagonica. This suggests that this coral is simply an opportunistic colonizer of free space made available by coastal habitat modifications. Our results suggest that unique genetic variation, possibly due to limited dispersal across the Ibiza Channel, an influx of individuals from different depths and/or adaptation to cooler temperatures along the northern expansion front may have facilitated the northward range expansion of O. patagonica in the western Mediterranean.

Microbial Diversity and Putative Diazotrophy in High- and Low-Microbial-Abundance Mediterranean Sponges.

Microbial communities associated with marine sponges carry out nutrient transformations essential for benthic-pelagic coupling; however, knowledge about their composition and function is still sparse. We evaluated the richness and diversity of prokaryotic assemblages associated with three high-microbial-abundance (HMA) and three low-microbial-abundance (LMA) sympatric Mediterranean sponges to address their stability and uniqueness. Moreover, to examine functionality and because an imbalance between nitrogen ingestion and excretion has been observed for some of these species, we sequenced nitrogenase genes (nifH) and measured N2 fixation. The prokaryotic communities in the two sponge types did not differ in terms of richness, but the highest diversity was found in HMA sponges. Moreover, the discrete composition of the communities in the two sponge types relative to that in the surrounding seawater indicated that horizontal transmission and vertical transmission affect the microbiomes associated with the two sponge categories. nifH genes were found in all LMA species and sporadically in one HMA species, and about half of the nifH gene sequences were common between the different sponge species and were also found in the surrounding water, suggesting horizontal transmission. (15)N2-enriched incubations showed that N2 fixation was measurable in the water but was not associated with the sponges. Also, the analysis of the isotopic ratio of (15)N to (14)N in sponge tissue indicated that N2 fixation is not an important source of nitrogen in these Mediterranean sponges. Overall, our results suggest that compositional and functional features differ between the prokaryotic communities associated with HMA and LMA sponges, which may affect sponge ecology.

Heterotrophy in marine animal forests in an era of climate change.

Marine animal forests (MAFs) are benthic ecosystems characterised by biogenic three-dimensional structures formed by suspension feeders such as corals, gorgonians, sponges and bivalves. They comprise highly diversified communities among the most productive in the world's oceans. However, MAFs are in decline due to global and local stressors that threaten the survival and growth of their foundational species and associated biodiversity. Innovative and scalable interventions are needed to address the degradation of MAFs and increase their resilience under global change. Surprisingly, few studies have considered trophic interactions and heterotrophic feeding of MAF suspension feeders as an integral component of MAF conservation. Yet, trophic interactions are important for nutrient cycling, energy flow within the food web, biodiversity, carbon sequestration, and MAF stability. This comprehensive review describes trophic interactions at all levels of ecological organisation in tropical, temperate, and cold-water MAFs. It examines the strengths and weaknesses of available tools for estimating the heterotrophic capacities of the foundational species in MAFs. It then discusses the threats that climate change poses to heterotrophic processes. Finally, it presents strategies for improving trophic interactions and heterotrophy, which can help to maintain the health and resilience of MAFs.

Specificity and temporal dynamics of complex bacteria--sponge symbiotic interactions.

Microbes are known to form intricate and intimate relationships with most animal and plant taxa. Microbe--host symbiotic associations are poorly explored in comparison with other species interaction networks. The current paradigm on symbiosis research stems from species-poor systems where pairwise and reciprocally specialized interactions between a single microbe and a single host that coevolve are the norm. These symbioses involving just a few species are fascinating in their own right, but more diverse and complex host-associated microbial communities are increasingly found, with new emerging questions that require new paradigms and approaches. Here we adopt an intermediate complexity approach to study the specificity, phylogenetic community structure, and temporal variability of the subset of the most abundant bacteria associated with different sponge host species with diverse eco-evolutionary characteristics. We do so by using a monthly resolved annual temporal series of host-associated and free-living bacteria. Bacteria are very abundant and diverse within marine sponges, and these symbiotic interactions are hypothesized to have a very ancient origin. We show that host-bacteria reciprocal specialization depends on the temporal scale and level of taxonomic aggregation considered. Sponge hosts with similar eco-evolutionary characteristics (e.g., volume of tissue corresponding to microbes, water filtering rates, and microbial transmission type) have similar bacterial phylogenetic community structure when looking at interactions aggregated over time. In general, sponge hosts hypothesized to form more intricate relationships with bacteria show a remarkably persistent bacterial community over time. Other hosts, however, show a large turnover similar to that observed for free-living bacterioplankton. Our study highlights the importance of exploring temporal variability in host--microbe interaction networks if we aim to determine how specific and persistent these poorly explored but extremely common interactions are.

Seroprevalence and socioeconomic impact of the first SARS-CoV-2 infection wave in a small town in Navarre, Spain.

The characterization of the antibody response to SARS-CoV-2 and its determinants are key for the understanding of COVID-19. The identification of vulnerable populations to the infection and to its socioeconomic impact is indispensable for inclusive policies. We conducted an age-stratified cross-sectional community-based seroprevalence survey between June 12th and 19th 2020-during the easing of lockdown-in Cizur, Spain. We quantified IgG, IgM and IgA levels against SARS-CoV-2 spike and its receptor-binding domain in a sample of 728 randomly selected, voluntarily registered inhabitants. We estimated a 7.9% seroprevalence in the general population, with the lowest seroprevalence among children under ten (n = 3/142, 2.1%) and the highest among adolescents (11-20 years old, n = 18/159, 11.3%). We found a heterogeneous immune-response profile across participants regarding isotype/antigen-specific seropositivity, although levels generally correlated. Those with technical education level were the most financially affected. Fifty-five percent had visited a supermarket and 43% a sanitary centre since mid-February 2020. When comparing by gender, men had left the household more frequently. In conclusion, few days after strict lockdown, the burden of SARS-CoV-2 infection was the lowest in children under 10. The findings also suggest that a wider isotype-antigen panel confers higher sensitivity. Finally, the economic impact biases should be considered when designing public health measures.

Familial CD45RA- T cells to treat severe refractory infections in immunocompromised patients.

Background: Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. Most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. The transfer of pathogen-specific Cytotoxic T-Lymphocytes has shown a minimal toxicity profile and effectiveness in treating Cytomegalovirus, Adenovirus, Epstein - Barr virus, BK Virus and Aspergillus infections, but this therapy have the main limitations of regulatory issues, high cost, and absence of public cell banks. However, CD45RA- cells containing pathogen-specific memory T-cells involve a less complex manufacturing and regulatory process and are cheaper, feasible, safe, and potentially effective. Methods: We present preliminary data from six immunocompromised patients: four who had severe infectious diseases and two who had EBV lymphoproliferative disease. All of them underwent multiple safe familial CD45RA- T-cell infusions as adoptive passive cell therapy, containing Cytomegalovirus, Epstein - Barr virus, BK virus, and Aspergillus-specific memory T-cells. We also present the method for selecting the best donors for CD45RA- cells in each case and the procedure to isolate and store these cells. Results: The infusions were safe, there was no case of graft-versus host disease, and they showed a clear clinical benefit. The patients treated for BK virus nephritis, Cytomegalovirus encephalitis, Cytomegalovirus reactivation, and disseminated invasive aspergillosis experienced pathogen clearance, complete resolution of symptoms in 4-6 weeks and a lymphocyte increase in 3 of 4 cases after 3-4 months. Donor T cell transient microchimerism was detected in one patient. The two patients treated for EBV lymphoproliferative disease underwent chemotherapy and several infusions of CD45RA- memory T-cells containing EBV cytotoxic lymphocytes. Donor T-cell microchimerism was observed in both patients. The viremia cleared in one of the patients, and in the other, despite the viremia not clearing, hepatic lymphoproliferative disease remained stable and was ultimately cured with EBV-specific Cytotoxic T-Lymphocytes. Conclusion: The use of familial CD45RA- T-cells containing specific Cytotoxic T-lymphocytes is a feasible, safe and potential effective approach for treating severe pathogen infections in immunocompromised patients through a third party donor. Furthermore, this approach might be of universal use with fewer institutional and regulatory barriers.

Imaging and Molecular Annotation of Xenographs and Tumours (IMAXT): High throughput data and analysis infrastructure.

With the aim of producing a 3D representation of tumors, imaging and molecular annotation of xenografts and tumors (IMAXT) uses a large variety of modalities in order to acquire tumor samples and produce a map of every cell in the tumor and its host environment. With the large volume and variety of data produced in the project, we developed automatic data workflows and analysis pipelines. We introduce a research methodology where scientists connect to a cloud environment to perform analysis close to where data are located, instead of bringing data to their local computers. Here, we present the data and analysis infrastructure, discuss the unique computational challenges and describe the analysis chains developed and deployed to generate molecularly annotated tumor models. Registration is achieved by use of a novel technique involving spherical fiducial marks that are visible in all imaging modalities used within IMAXT. The automatic pipelines are highly optimized and allow to obtain processed datasets several times quicker than current solutions narrowing the gap between data acquisition and scientific exploitation.

Nutrient fluxes through sponges: biology, budgets, and ecological implications.

Marine sponges are able to process a variety of carbon (C), nitrogen (N), phosphorous (P), and silicon (Si) dissolved compounds, in addition to the particulate C, N, and P obtained through regular feeding. While Si fluxes through sponges are exclusively related to the elaboration of their skeleton of biogenic silica, C, N, and P fluxes derive from a complex combination of metabolic processes that include feeding, respiration, egestion, excretion, as well as hosting of large microbial populations within the sponge body. Because of the remarkable abundance of sponges in many benthic marine communities, they have the potential to impact the availability of the compounds they take up and release, affecting the benthic-pelagic coupling and cycling rates of chemical elements that are crucial to determine growth of bacterioplankton and primary producers at the ecosystem level. Unfortunately, our knowledge and understanding of the magnitude of the sponge-meditated nutrient fluxes and their ecological implications depends much on the compound type (i.e. C, N, P, or Si). Herein, we review the available knowledge on the subject with emphasis on recent developments.

Global warming-enhanced stratification and mass mortality events in the Mediterranean.

Summer conditions in the Mediterranean Sea are characterized by high temperatures and low food availability. This leads to "summer dormancy" in many benthic suspension feeders due to energetic constraints. Analysis of the most recent 33-year temperature time series demonstrated enhanced stratification due to global warming, which produced a approximately 40% lengthening of summer conditions. Many biological processes are expected to be affected by this trend, culminating in such events as mass mortality of invertebrates. Climatic anomalies concomitant with the occurrence of these events represent prolonged exposure to warmer summer conditions coupled with reduced food resources. Simulation of the effects of these conditions on a model organism demonstrated a biomass loss of >35%. Losses of this magnitude result in mortality similar to that noted in field observations during mass mortality events. These results indicate that temperature anomalies are the underlying cause of the events, with energetic constraints serving as the main triggering mechanism.

Wounding response in Porifera (sponges) activates ancestral signaling cascades involved in animal healing, regeneration, and cancer.

Upon injury, the homeostatic balance that ensures tissue function is disrupted. Wound-induced signaling triggers the recovery of tissue integrity and offers a context to understand the molecular mechanisms for restoring tissue homeostasis upon disturbances. Marine sessile animals are particularly vulnerable to chronic wounds caused by grazers that can compromise prey's health. Yet, in comparison to other stressors like warming or acidification, we know little on how marine animals respond to grazing. Marine sponges (Phylum Porifera) are among the earliest-diverging animals and play key roles in the ecosystem; but they remain largely understudied. Here, we investigated the transcriptomic responses to injury caused by a specialist spongivorous opisthobranch (i.e., grazing treatment) or by clipping with a scalpel (i.e., mechanical damage treatment), in comparison to control sponges. We collected samples 3 h, 1 d, and 6 d post-treatment for differential gene expression analysis on RNA-seq data. Both grazing and mechanical damage activated a similar transcriptomic response, including a clotting-like cascade (e.g., with genes annotated as transglutaminases, metalloproteases, and integrins), calcium signaling, and Wnt and mitogen-activated protein kinase signaling pathways. Wound-induced gene expression signature in sponges resembles the initial steps of whole-body regeneration in other animals. Also, the set of genes responding to wounding in sponges included putative orthologs of cancer-related human genes. Further insights can be gained from taking sponge wound healing as an experimental system to understand how ancient genes and regulatory networks determine healthy animal tissues.

Resolvin E1 attenuates doxorubicin-induced endothelial senescence by modulating NLRP3 inflammasome activation.

Endothelial cell senescence contributes to chronic inflammation and endothelial dysfunction, while favoring cardiovascular disorders and frailty. Senescent cells acquire a pro-inflammatory secretory phenotype that further propagates inflammation and senescence to neighboring cells. Cell senescence can be provoked by plethora of stressors, including inflammatory molecules and chemotherapeutic drugs. Doxorubicin (Doxo) is a powerful anthracycline anticancer drug whose clinical application is constrained by a dose-limiting cardiovascular toxicity. We here investigated whether cell senescence can contribute to the vascular damage elicited by Doxo. In human umbilical vein endothelial cells (HUVEC) cultures, Doxo (10-100 nM) increased the number of SA-ß-gal positive cells and the levels of γH2AX, p21 and p53, used as markers of senescence. Moreover, we identified Doxo-induced senescence to be mediated by the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, a key player of the immune innate system capable of releasing interleukin (IL)-1ß. In fact, IL-1ß itself mimicked the stimulatory action of Doxo on both NLRP3 activation and cellular senescence, while the pharmacological blockade of IL-1 receptors markedly attenuated the pro-senescence effects of Doxo. In search of additional pharmacological strategies to attenuate Doxo-induced endothelial senescence, we identified resolvin E1 (RvE1), an endogenous pro-resolving mediator, as capable of reducing cell senescence induced by both Doxo and IL-1ß by interfering with the increased expression of pP65, NLRP3, and pro-IL-1ß proteins and with the formation of active NLRP3 inflammasome complexes. Overall, RvE1 and the blockade of the NLRP3 inflammasome-IL-1ß axis may offer a novel therapeutic approach against Doxo-induced cardiovascular toxicity and subsequent sequelae.

Determinants of early antibody responses to COVID-19 mRNA vaccines in a cohort of exposed and naïve healthcare workers.

BACKGROUND: Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. METHODS: We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. FINDINGS: Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p<0·05). A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with 43% (95% CI, 19-59) and 45% (95% CI, 63-18) lower neutralization, respectively, and 35% (95% CI, 3-57%) and 55% (95% CI, 33-70%) lower antibody levels, respectively. Among fully vaccinated, 6·3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. INTERPRETATION: Our data support administering a single-dose in pre-exposed healthy individuals as primary vaccination. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination and in face of variants that escape immunity such as Omicron. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FUNDING: This work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

The NALCN channel regulates metastasis and nonmalignant cell dissemination.

We identify the sodium leak channel non-selective protein (NALCN) as a key regulator of cancer metastasis and nonmalignant cell dissemination. Among 10,022 human cancers, NALCN loss-of-function mutations were enriched in gastric and colorectal cancers. Deletion of Nalcn from gastric, intestinal or pancreatic adenocarcinomas in mice did not alter tumor incidence, but markedly increased the number of circulating tumor cells (CTCs) and metastases. Treatment of these mice with gadolinium-a NALCN channel blocker-similarly increased CTCs and metastases. Deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer caused shedding of epithelial cells into the blood at levels equivalent to those seen in tumor-bearing animals. These cells trafficked to distant organs to form normal structures including lung epithelium, and kidney glomeruli and tubules. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumorigenesis and unmasking a potential new target for antimetastatic therapies.