Sympathovagal quotient and resting-state functional connectivity of control networks are related to gut Ruminococcaceae abundance in healthy men.

INTRODUCTION: Heart rate variability (HRV), brain resting-state functional connectivity (rsFC), and gut microbiota (GM) are three recognized indicators of health status, whose relationship has not been characterized. We aimed to identify the GM genera and families related to HRV and rsFC, the interaction effect of HRV and rsFC on GM taxa abundance, and the mediation effect of diet on these relationships. METHODS: Eighty-eight healthy, young Colombian men were included in this cross-sectional study. HRV metrics were extracted from 24-hour Holter monitoring data and the resting functional connectivity strength (FCS) of 15 networks were derived from functional magnetic resonance imaging. Gut microbiota composition was assessed using the sequences of the V3-V4 regions of the 16 S rRNA gene, and diet was evaluated using a food frequency questionnaire. Multivariate linear regression analyses were performed to evaluate the correlations between the independent variables (HRV metrics and FCS) and the dependent variables (GM taxa abundance or alpha diversity indexes). Mediation analyses were used to test the role of diet in the relationship between HRV and GM. RESULTS: The sympathovagal quotient (SQ) and the FCS of control networks were positively correlated with the abundance of the gut Ruminococcaceae family and an unclassified Ruminococcaceae genus (Ruminococcaceae_unc). Additionally, the interaction between the FCS of the control network and SQ reduced the individual main effects on the Ruminococcaceae_unc abundance. Finally, reduced habitual fiber intake partially mediated the relationship between SQ and this genus. CONCLUSION: Two indicators of self-regulation, HRV and the rsFC of control networks, are related to the abundance of gut microbiota taxa in healthy men. However, only HRV is related to habitual dietary intake; thus, HRV could serve as a marker of food choice and GM composition in the future.

Lipin-2 regulates the antiviral and anti-inflammatory responses to interferon.

Interferons (IFN) are crucial antiviral and immunomodulatory cytokines that exert their function through the regulation of a myriad of genes, many of which are not yet characterized. Here, we reveal that lipin-2, a phosphatidic acid phosphatase whose mutations produce an autoinflammatory syndrome known as Majeed syndrome in humans, is regulated by IFN in a STAT-1-dependent manner. Lipin-2 inhibits viral replication both in vitro and in vivo. Moreover, lipin-2 also acts as a regulator of inflammation in a viral context by reducing the signaling through TLR3 and the generation of ROS and release of mtDNA that ultimately activate the NLRP3 inflammasome. Inhibitors of mtDNA release from mitochondria restrict IL-1ß production in lipin-2-deficient animals in a model of viral infection. Finally, analyses of databases from COVID-19 patients show that LPIN2 expression levels negatively correlate with the severity of the disease. Overall, these results uncover novel regulatory mechanisms of the IFN response driven by lipin-2 and open new perspectives for the future management of patients with LPIN2 mutations.

Axillary Cutaneous Hemosiderosis in a Patient With Hyperhidrosis, After Intravenous Iron Infusion.

ABSTRACT: Hemosiderosis consists of an iron deposit in tissues, which does not cause organic damage to them. However, in the case of the skin, being an organ exposed to sight, siderosis can produce a brownish coloration that is aesthetically discomforting for the individual. Most cutaneous sideroses are because of venous insufficiency with hemorrhagic extravasation. There is also a group secondary to iron extravasation in the injection site of the transfusion. However, there are very few cases in which an intravenous injection of an iron preparation has produced diffuse siderosis in extensive areas of the skin. We present the case of a 31-year-old woman with hyperhidrosis, who was transfused as a result of postpartum hemorrhage and, shortly after receiving an intravenous iron infusion, developed extensive hyperpigmentation in both axillae.

Response to Neoadjuvant Therapy and Long-Term Survival in Patients With Triple-Negative Breast Cancer.

PURPOSE: Triple-negative breast cancer (TNBC) is defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) expression. In this study, we compared response to neoadjuvant chemotherapy and survival between patients with TNBC and non-TNBC. PATIENTS AND METHODS: Analysis of a prospectively collected clinical database was performed. We included 1,118 patients who received neoadjuvant chemotherapy at M.D. Anderson Cancer Center for stage I-III breast cancer from 1985 to 2004 and for whom complete receptor information were available. Clinical and pathologic parameters, pathologic complete response rates (pCR), survival measurements, and organ-specific relapse rates were compared between patients with TNBC and non-TNBC. RESULTS: Two hundred fifty-five patients (23%) had TNBC. Patients with TNBC compared with non-TNBC had significantly higher pCR rates (22% v 11%; P = .034), but decreased 3-year progression-free survival rates (P < .0001) and 3-year overall survival (OS) rates (P < .0001). TNBC was associated with increased risk for visceral metastases (P = .0005), lower risk for bone recurrence (P = .027), and shorter postrecurrence survival (P < .0001). Recurrence and death rates were higher for TNBC only in the first 3 years. If pCR was achieved, patients with TNBC and non-TNBC had similar survival (P = .24). In contrast, patients with residual disease (RD) had worse OS if they had TNBC compared with non-TNBC (P < .0001). CONCLUSION: Patients with TNBC have increased pCR rates compared with non-TNBC, and those with pCR have excellent survival. However, patients with RD after neoadjuvant chemotherapy have significantly worse survival if they have TNBC compared with non-TNBC, particularly in the first 3 years.

IDENTIFICATION OF ANTHROPOGENIC IMPACT ON NATURAL HABITATS BY ANTIMICROBIAL RESISTANCE QUANTIFICATION IN TWO NEOTROPICAL WILD CATS AND THEIR GEOSPATIAL ANALYSIS.

Human activities such as habitat degradation and fragmentation threaten biodiversity in Neotropical areas. This work proposes an analytical methodology to identify natural areas in Central America with anthropogenic impact, analyzing the presence of antimicrobial resistance genes (ARGs) in accordance with their theoretical relationship with human-related activities. Sixteen ARGs were quantified in feces of different individuals of 13 jaguars (Panthera onca) and 13 pumas (Puma concolor) in three conservation areas in Costa Rica by real-time PCR. At least one ARG was detected in all samples. Of the ARGs encoding tetracycline resistance, the most frequent were tetQ and tetY (85% and 69%, respectively). The sulfonamides (sulI and sulII; 69% each), phenicols (catI and catII; 19% and 54%, respectively), and quinolones (qnrS; 12%) were also detected. The presence of human settlements, livestock farms (pigs, cattle, and poultry), roads, human health centers, flood zones, and rivers were identified within each area to generate an index of human activity. We found no difference between the presence of ARG by roads, agricultural activities, and human settlements (P>0.05). However, tetW showed higher percentages with porcine and bovine farms; both tetY and tetW were more frequent in jaguars than in pumas. Of concern is that many of the most contaminated samples were taken from national parks, such as Braulio Carrillo and Tortuguero, where animals should not have direct contact with humans.

RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers.

The ability of antibodies to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important correlate of protection. For routine evaluation of protection, however, a simple and cost-efficient anti-SARS-CoV-2 serological assay predictive of serum neutralizing activity is needed. We analyzed clinical epidemiological data and blood samples from two cohorts of health care workers in Barcelona and Munich to compare several immunological readouts for evaluating antibody levels that could be surrogates of neutralizing activity. We measured IgG levels against SARS-CoV-2 spike protein (S), its S2 subunit, the S1 receptor binding domain (RBD), and the full length and C terminus of nucleocapsid (N) protein by Luminex, and against RBD by enzyme-linked immunosorbent assay (ELISA), and assessed those as predictors of plasma surrogate-neutralizing activity measured by a flow cytometry assay. In addition, we determined the clinical and demographic factors affecting plasma surrogate-neutralizing capacity. Both cohorts showed a high positive correlation between IgG levels to S antigen, especially to RBD, and the levels of plasma surrogate-neutralizing activity, suggesting RBD IgG as a good correlate of plasma neutralizing activity. Symptomatic infection, with symptoms such as loss of taste, dyspnea, rigors, fever and fatigue, was positively associated with anti-RBD IgG positivity by ELISA and Luminex, and with plasma surrogate-neutralizing activity. Our serological assays allow for the prediction of serum neutralization activity without the cost, hazards, time, and expertise needed for surrogate or conventional neutralization assays. Once a cutoff is established, these relatively simple high-throughput antibody assays will provide a fast and cost-effective method of assessing levels of protection from SARS-CoV-2 infection. IMPORTANCE Neutralizing antibody titers are the best correlate of protection against SARS-CoV-2. However, current tests to measure plasma or serum neutralizing activity do not allow high-throughput screening at the population level. Serological tests could be an alternative if they are proved to be good predictors of plasma neutralizing activity. In this study, we analyzed the SARS-CoV-2 serological profiles of two cohorts of health care workers by applying Luminex and ELISA in-house serological assays. Correlations of both serological tests were assessed between them and with a flow cytometry assay to determine plasma surrogate-neutralizing activity. Both assays showed a high positive correlation between IgG levels to S antigens, especially RBD, and the levels of plasma surrogate-neutralizing activity. This result suggests IgG to RBD as a good correlate of plasma surrogate-neutralizing activity and indicates that serology of IgG to RBD could be used to assess levels of protection from SARS-CoV-2 infection.

Discovery of the first PD-1 ligand encoded by a pathogen.

Large double-stranded DNA viruses deploy multiple strategies to subvert host immune defenses. Some of these tactics are mediated by viral gene products acquired by horizontal gene transfer from the corresponding hosts and shaped throughout evolution. The programmed death-1 (PD-1) receptor and its ligands, PD-L1 and PD-L2, play a pivotal role attenuating T-cell responses and regulating immune tolerance. In this study, we report the first functional PD-L1 homolog gene (De2) found in a pathogen. De2, captured by a γ-herpesvirus from its host during co-evolution around 50 million years ago, encodes a cell-surface glycoprotein that interacts with high affinity and stability with host PD-1. We also find that mutations evolved by the viral protein result in a significant loss of its ability to interact in cis with CD80, an interaction that for PD-L1:CD80 has been reported to block PD-1 inhibitory pathways. Furthermore, we demonstrate that the viral protein strongly inhibits T-cell signaling. Our observations suggest that PD-L1 homologs may enable viruses to evade T cell responses, favor their replication, and prevent excessive tissue damage. Altogether, our findings reveal a novel viral immunosuppressive strategy and highlight the importance of the modulation of the PD-1/PD-L1 axis during viral infections.

Decreased and Heterogeneous Neutralizing Antibody Responses Against RBD of SARS-CoV-2 Variants After mRNA Vaccination.

The rapid spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerging variants raises concerns about their capacity to evade immune protection provided by natural infection or vaccination. The receptor-binding domain (RBD) of the viral spike protein is the major target of neutralizing antibodies, and viral variants accumulate mutations in this region. In this study, we determined the antibody neutralization capacity against the RBD of SARS-CoV-2 variants Alpha (B.1.1.7), Gamma (P.1), Epsilon (B.1.427), Kappa (B.1.617.1), and Delta (B.1.617.2) in a cohort of healthcare workers naturally infected or receiving COVID-19 mRNA vaccines from Moderna or Pfizer-BioNTech. We show that the five RBD variants displayed an augmented binding to ACE2 compared to the original Wuhan strain. The most significant increase was observed in variants Epsilon and Delta, containing mutation L452R. Using a flow cytometry cell-based assay, we found that SARS-CoV-2-infected subjects presented low levels of RBD-specific neutralizing antibodies against all variants analyzed, except Alpha. However, the neutralizing activity incremented considerably after a subsequent mRNA-vaccine dose, to levels significantly higher than those in naïve individuals receiving two vaccine doses. Importantly, we observed partially impaired neutralizing responses against most variants in fully vaccinated individuals. Variants Gamma and Kappa encompassing RBD E484K/Q mutations presented the highest neutralizing resistance. Furthermore, a wide heterogeneity in the magnitude of RBD-specific neutralizing responses against all tested SARS-CoV-2 variants following both mRNA vaccines was detected. Altogether, our findings provide important knowledge regarding SARS-CoV-2 vaccine-induced immunity, and should be very useful to guide future vaccination regimens and personalized vaccine approaches.

Spineless Cactus plus Urea and Tifton-85 Hay: Maximizing the Digestible Organic Matter Intake, Ruminal Fermentation and Nitrogen Utilization of Wethers in Semi-Arid Regions.

The aim of this study was to evaluate the effect of replacing Tifton-85 hay (Cynodon spp. cv. Tifton 85) with 0, 150, 300, 450 and 600 g/kg dry matter (DM) of spineless cactus (SC, Nopalea cochenilifera Salm-Dyck) plus urea and ammonium sulfate (UAS; 9:1) on DM, digestible organic matter (DOM) and indigestible neutral detergent fiber (iNDF) intakes, as well as ruminal fermentation, N-balance, and microbial protein supply (MPS). Five rumen-fistulated and cannulated crossbred wethers, weighing 43.8 ± 5.80 kg, were randomized in a 5 × 5 Latin square design. Isonitrogenous diets (14% crude protein) were supplied with a roughage/concentrate ratio of 70:30. The DOM intake, N-retained, and MPS showed quadratic responses (p < 0.05), with maximum values estimated at the levels of SC+UAS of 414, 438 and 418 g/kg DM, respectively. Rumen pH and ammonia nitrogen, iNDF intake, N-urinary excretion, and serum urea and plasma ammonia reduced linearly (p < 0.05) with increasing SC+UAS inclusion. Ruminal acetate and propionate concentrations increased linearly with increasing SC+UAS inclusion. In wethers fed diets with a roughage/concentrate ratio of 70:30, roughage constituted of a SC+UAS/hay (Tifton-85) ratio of 41:29 is recommended in order to maximize the DOM intake, N-retention, and MPS.

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.

Factores de riesgo de fracturas no vertebrales en receptores de trasplante renal: ¿Una realidad compleja? / Risk factors for nonvertebral bone fractures in kidney transplant recipients: a complex reality?

RESUMEN Introducción: Las fracturas en el postrasplante renal representan una de las complicaciones que aumentan la morbimortalidad en los pacientes. Objetivo: Determinar los factores de riesgo de trasplante de fracturas no vertebrales en receptores de trasplante renal. Material y métodos: Se analizaron los datos de historias clínicas de 737 pacientes trasplantados renales desde 2009-2015, donde se evaluaron los factores de riesgo de fracturas. Se determinaron como factores generales: edad, sexo; y factores específicos: etiología de la enfermedad renal crónica (ERC), tiempo en diálisis, modalidad de diálisis, características del donante:edad, sexo, tipo de donante vivo y cadavérico (DV, DC), diferentes esquemas de inmunosupresión, rechazos, tratamiento con pulsos de esteroides, uso de bifosfonatos y analitos como parathormona intacta (PTHi), pro-vitamina D (25OHD) y creatininemia (Creats). Criterios de exclusión: pacientes <18 años, retrasplante o trasplantes dobles y menos de 1 año de seguimiento. Resultados: 44 pacientes presentaron 50 fracturas no vertebrales. El 62% de las fracturas se presentaron en los 3 primeros años del trasplante. En el modelo Multivariado se pudo observar que los factores de riesgo generales asociados con riesgo de fracturas fueron la edad avanzada del receptor (aHR por año aumenta 1,03, 95% CI: 1,00-1,05] y el sexo femenino (aHR= 1,60, 95% CI: 0,85-2,99), mientras que los factores de riesgo específicos fueron el tiempo en diálisis (1,07 IC95%:1,02- 1,12), la mayor edad del donante (1,03 IC95:1,00- 1,05) y el déficit de 25OHD (3,17 IC95:1,57- 6,41). Conclusiones: Este estudio permitió evaluar los factores predictivos de fractura en nuestros pacientes con trasplante renal.

ABSTRACT Introduction: Fractures in renal postransplantation represent one of the complications that increase morbidity and mortality in patients. Objective: To determine the risk factors for transplantation of non- vertebral fractures in renal transplant recipients. Methods: The data of clinical histories of 737 renal transplanted patients since 2009 2015 were analyzed, where the risk factors of fractures were evaluated. They were determined as general factors: age, sex; and specific factors: etiology of chronic kidney disease (ERC), dialysis time, dialysis modality, donor characteristics: age, sex, type of donor, living or cadaveric (DV, DC), different immunosuppression treatments, rejections, treatment with steroid pulses, use of bisphosphonates and biochemical parameters like intact parathormone (PTHI), Pro-Vitamin D (25OHD) and Creatininemia (CREATS). Exclusión criteria: patients <18 years, retransplantation or double transplants and follow- up less than 1 year. Results: 44 patients presented 50 non-vertebral fractures. 62% of the fractures were presented in the first 3 years of the transplant. In the multivariate model it was observed that the general risk factors associated with fracture risk were the advanced age of the receptor (aHR per year increases 1.03, 95% CI: 1.00-1.05] and the female sex (aHR = 1.60, 95% CI: 0.85-2.99), while the specific risk factors were dialysis time (1.07 IC95%: 1.02-1.12), the higher age of the donor (1.03 IC95: 1,00-1,05) and the deficit of 25OHD (3.17 IC95: 1.57-6.41). Conclusions: This study allowed to evaluate predictive factors of fracture in our patients with kidney transplantation.

Oxidative Phosphorylation Is a Metabolic Vulnerability in Chemotherapy-Resistant Triple-Negative Breast Cancer.

Oxidative phosphorylation (OXPHOS) is an active metabolic pathway in many cancers. RNA from pretreatment biopsies from patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy demonstrated that the top canonical pathway associated with worse outcome was higher expression of OXPHOS signature. IACS-10759, a novel inhibitor of OXPHOS, stabilized growth in multiple TNBC patient-derived xenografts (PDX). On gene expression profiling, all of the sensitive models displayed a basal-like 1 TNBC subtype. Expression of mitochondrial genes was significantly higher in sensitive PDXs. An in vivo functional genomics screen to identify synthetic lethal targets in tumors treated with IACS-10759 found several potential targets, including CDK4. We validated the antitumor efficacy of the combination of palbociclib, a CDK4/6 inhibitor, and IACS-10759 in vitro and in vivo. In addition, the combination of IACS-10759 and multikinase inhibitor cabozantinib had improved antitumor efficacy. Taken together, our data suggest that OXPHOS is a metabolic vulnerability in TNBC that may be leveraged with novel therapeutics in combination regimens. SIGNIFICANCE: These findings suggest that triple-negative breast cancer is highly reliant on OXPHOS and that inhibiting OXPHOS may be a novel approach to enhance efficacy of several targeted therapies.

Seven-month kinetics of SARS-CoV-2 antibodies and role of pre-existing antibodies to human coronaviruses.

Unraveling the long-term kinetics of antibodies to SARS-CoV-2 and the individual characteristics influencing it, including the impact of pre-existing antibodies to human coronaviruses causing common cold (HCoVs), is essential to understand protective immunity to COVID-19 and devise effective surveillance strategies. IgM, IgA and IgG levels against six SARS-CoV-2 antigens and the nucleocapsid antigen of the four HCoV (229E, NL63, OC43 and HKU1) were quantified by Luminex, and antibody neutralization capacity was assessed by flow cytometry, in a cohort of health care workers followed up to 7 months (N = 578). Seroprevalence increases over time from 13.5% (month 0) and 15.6% (month 1) to 16.4% (month 6). Levels of antibodies, including those with neutralizing capacity, are stable over time, except IgG to nucleocapsid antigen and IgM levels that wane. After the peak response, anti-spike antibody levels increase from ~150 days post-symptom onset in all individuals (73% for IgG), in the absence of any evidence of re-exposure. IgG and IgA to HCoV are significantly higher in asymptomatic than symptomatic seropositive individuals. Thus, pre-existing cross-reactive HCoVs antibodies could have a protective effect against SARS-CoV-2 infection and COVID-19 disease.

Genomic, Transcriptomic, and Proteomic Profiling of Metastatic Breast Cancer.

PURPOSE: Metastatic breast cancer (MBC) is not curable and there is a growing interest in personalized therapy options. Here we report molecular profiling of MBC focusing on molecular evolution in actionable alterations. EXPERIMENTAL DESIGN: Sixty-two patients with MBC were included. An analysis of DNA, RNA, and functional proteomics was done, and matched primary and metastatic tumors were compared when feasible. RESULTS: Targeted exome sequencing of 41 tumors identified common alterations in TP53 (21; 51%) and PIK3CA (20; 49%), as well as alterations in several emerging biomarkers such as NF1 mutations/deletions (6; 15%), PTEN mutations (4; 10%), and ARID1A mutations/deletions (6; 15%). Among 27 hormone receptor-positive patients, we identified MDM2 amplifications (3; 11%), FGFR1 amplifications (5; 19%), ATM mutations (2; 7%), and ESR1 mutations (4; 15%). In 10 patients with matched primary and metastatic tumors that underwent targeted exome sequencing, discordances in actionable alterations were common, including NF1 loss in 3 patients, loss of PIK3CA mutation in 1 patient, and acquired ESR1 mutations in 3 patients. RNA sequencing in matched samples confirmed loss of NF1 expression with genomic NF1 loss. Among 33 patients with matched primary and metastatic samples that underwent RNA profiling, 14 actionable genes were differentially expressed, including antibody-drug conjugate targets LIV-1 and B7-H3. CONCLUSIONS: Molecular profiling in MBC reveals multiple common as well as less frequent but potentially actionable alterations. Genomic and transcriptional profiling demonstrates intertumoral heterogeneity and potential evolution of actionable targets with tumor progression. Further work is needed to optimize testing and integrated analysis for treatment selection.

Cytomegalovirus restricts ICOSL expression on antigen-presenting cells disabling T cell co-stimulation and contributing to immune evasion.

Viral infections are controlled, and very often cleared, by activated T lymphocytes. The inducible co-stimulator (ICOS) mediates its functions by binding to its ligand ICOSL, enhancing T-cell activation and optimal germinal center (GC) formation. Here, we show that ICOSL is heavily downmodulated during infection of antigen-presenting cells by different herpesviruses. We found that, in murine cytomegalovirus (MCMV), the immunoevasin m138/fcr-1 physically interacts with ICOSL, impeding its maturation and promoting its lysosomal degradation. This viral protein counteracts T-cell responses, in an ICOS-dependent manner, and limits virus control during the acute MCMV infection. Additionally, we report that blockade of ICOSL in MCMV-infected mice critically regulates the production of MCMV-specific antibodies due to a reduction of T follicular helper and GC B cells. Altogether, these findings reveal a novel mechanism evolved by MCMV to counteract adaptive immune surveillance, and demonstrates a role of the ICOS:ICOSL axis in the host defense against herpesviruses.

Divergent Traits and Ligand-Binding Properties of the Cytomegalovirus CD48 Gene Family.

The genesis of gene families by the capture of host genes and their subsequent duplication is a crucial process in the evolution of large DNA viruses. CD48 is a cell surface molecule that interacts via its N-terminal immunoglobulin (Ig) domain with the cell surface receptor 2B4 (CD244), regulating leukocyte cytotoxicity. We previously reported the presence of five CD48 homologs (vCD48s) in two related cytomegaloviruses, and demonstrated that one of them, A43, binds 2B4 and acts as a soluble CD48 decoy receptor impairing NK cell function. Here, we have characterized the rest of these vCD48s. We show that they are highly glycosylated proteins that display remarkably distinct features: divergent biochemical properties, cellular locations, and temporal expression kinetics. In contrast to A43, none of them interacts with 2B4. Consistent with this, molecular modeling of the N-terminal Ig domains of these vCD48s evidences notable changes as compared to CD48, suggesting that they interact with alternative targets. Accordingly, we demonstrate that one of them, S30, tightly binds CD2, a crucial T- and NK-cell adhesion and costimulatory molecule. Thus, our findings show how a key host immune receptor gene captured by a virus can be subsequently remodeled to evolve new immunoevasins with altered binding properties.

Cytomegalovirus protein m154 perturbs the adaptor protein-1 compartment mediating broad-spectrum immune evasion.

Cytomegaloviruses (CMVs) are ubiquitous pathogens known to employ numerous immunoevasive strategies that significantly impair the ability of the immune system to eliminate the infected cells. Here, we report that the single mouse CMV (MCMV) protein, m154, downregulates multiple surface molecules involved in the activation and costimulation of the immune cells. We demonstrate that m154 uses its cytoplasmic tail motif, DD, to interfere with the adaptor protein-1 (AP-1) complex, implicated in intracellular protein sorting and packaging. As a consequence of the perturbed AP-1 sorting, m154 promotes lysosomal degradation of several proteins involved in T cell costimulation, thus impairing virus-specific CD8+ T cell response and virus control in vivo. Additionally, we show that HCMV infection similarly interferes with the AP-1 complex. Altogether, we identify the robust mechanism employed by single viral immunomodulatory protein targeting a broad spectrum of cell surface molecules involved in the antiviral immune response.

The transcription factor NFAT5 limits infection-induced type I interferon responses.

Type I interferon (IFN-I) provides effective antiviral immunity but can exacerbate harmful inflammatory reactions and cause hematopoietic stem cell (HSC) exhaustion; therefore, IFN-I expression must be tightly controlled. While signaling mechanisms that limit IFN-I induction and function have been extensively studied, less is known about transcriptional repressors acting directly on IFN-I regulatory regions. We show that NFAT5, an activator of macrophage pro-inflammatory responses, represses Toll-like receptor 3 and virus-induced expression of IFN-I in macrophages and dendritic cells. Mice lacking NFAT5 exhibit increased IFN-I production and better control of viral burden upon LCMV infection but show exacerbated HSC activation under systemic poly(I:C)-induced inflammation. We identify IFNß as a primary target repressed by NFAT5, which opposes the master IFN-I inducer IRF3 by binding to an evolutionarily conserved sequence in the IFNB1 enhanceosome that overlaps a key IRF site. These findings illustrate how IFN-I responses are balanced by simultaneously opposing transcription factors.

Dynamic clonal remodelling in breast cancer metastases is associated with subtype conversion.

BACKGROUND: Changes in the clinical subtype (CS) and intrinsic subtype (IS) between breast cancer (BC) metastases and corresponding primary tumours have been reported. However, their relationship with tumour genomic changes remains poorly characterised. Here, we analysed the association between genomic remodelling and subtype conversion in paired primary and metastatic BC samples. METHODS: A total of 57 paired primary and metastatic tumours from GEICAM/2009-03 (ConvertHER, NCT01377363) study participants with centrally assessed CS (n = 57) and IS (n = 46) were analysed. Targeted capture and next-generation sequencing of 202 genes on formalin-fixed paraffin-embedded samples was performed. The cancer cell fraction (CCF) of mutations in primary and metastatic pairs was estimated as a surrogate of tumour clonal architecture. Changes in mutation CCF between matched primary and metastatic tumours were analysed in the presence or absence of subtype conversion. FINDINGS: CS conversion occurred in 24.6% and IS conversion occurred in 36.9% of metastases. Primary tumours and metastases had a median of 11 (range, 3-29) and 9 (range, 1-38) mutations, respectively (P = 0.05). Overall, mutations in metastases showed a higher estimated CCF than in primary tumours (median CCF, 0.51 and 0.47, respectively; P = 0.042), consistent with increased clonal homogeneity. The increase in mutation CCF was significant in CS-converted (P = 0.04) but not in IS-converted (P = 0.48) metastases. Clonal remodelling was highest in metastases from hormone receptor-positive and human epidermal growth factor 2 (HER2)-positive tumours (P = 0.006). CONCLUSIONS: Mutations in BC metastases showed significantly higher estimated CCF than primary tumours. CCF changes were more prominent in metastases with CS conversion. Our findings suggest that changes in BC subtypes are linked to clonal remodelling during BC evolution.

Phase II trial of AKT inhibitor MK-2206 in patients with advanced breast cancer who have tumors with PIK3CA or AKT mutations, and/or PTEN loss/PTEN mutation.

BACKGROUND: The PI3K/AKT pathway is activated through PIK3CA or AKT1 mutations and PTEN loss in breast cancer. We conducted a phase II trial with an allosteric AKT inhibitor MK-2206 in patients with advanced breast cancer who had tumors with PIK3CA/AKT1 mutations and/or PTEN loss/mutation. METHODS: The primary endpoint was objective response rate (ORR). Secondary endpoints were 6-month progression-free survival (6 m PFS), predictive and pharmacodynamic markers, safety, and tolerability. Patients had pre-treatment and on-treatment biopsies as well as collection of peripheral blood mononuclear cells (PBMC) and platelet-rich plasma (PRP). Next-generation sequencing, immunohistochemistry, and reverse phase protein arrays (RPPA) were performed. RESULTS: Twenty-seven patients received MK-2206. Eighteen patients were enrolled into the PIK3CA/AKT1 mutation arm (cohort A): 13 had PIK3CA mutations, four had AKT1 mutations, and one had a PIK3CA mutation as well as PTEN loss. ORR and 6 m PFS were both 5.6% (1/18), with one patient with HR+ breast cancer and a PIK3CA E542K mutation experiencing a partial response (on treatment for 36 weeks). Nine patients were enrolled on the PTEN loss/mutation arm (cohort B). ORR was 0% and 6 m PFS was 11% (1/9), observed in a patient with triple-negative breast cancer and PTEN loss. The study was stopped early due to futility. The most common adverse events were fatigue (48%) and rash (44%). On pre-treatment biopsy, PIK3CA and AKT1 mutation status was concordant with archival tissue testing. However, two patients with PTEN loss based on archival testing had PTEN expression on the pre-treatment biopsy. MK-2206 treatment was associated with a significant decline in pAKT S473 and pAKT T308 and PI3K activation score in PBMC and PRPs, but not in tumor biopsies. By IHC, there was no significant decrease in median pAKT S473 or Ki-67 staining, but a drop was observed in both responders. CONCLUSIONS: MK-2206 monotherapy had limited clinical activity in advanced breast cancer patients selected for PIK3CA/AKT1 or PTEN mutations or PTEN loss. This may, in part, be due to inadequate target inhibition at tolerable doses in heavily pre-treated patients with pathway activation, as well as tumor heterogeneity and evolution in markers such as PTEN conferring challenges in patient selection. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01277757 . Registered 13 January 2011.