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RIFINs displayed on malaria-infected erythrocytes bind KIR2DL1 and KIR2DS1.
Natural killer (NK) cells use inhibitory and activating immune receptors to differentiate between human cells and pathogens. Signalling by these receptors determines whether an NK cell becomes activated and destroys a target cell. In some cases, such as killer immunoglobulin-like receptors, immune receptors are found in pairs, with inhibitory and activating receptors containing nearly identical extracellular ligand-binding domains coupled to different intracellular signalling domains1. Previous studies showed that repetitive interspersed family (RIFIN) proteins, displayed on the surfaces of Plasmodium falciparum-infected erythrocytes, can bind to inhibitory immune receptors and dampen NK cell activation2,3, reducing parasite killing. However, no pathogen-derived ligand has been identified for any human activating receptor. Here we identified a clade of RIFINs that bind to inhibitory immune receptor KIR2DL1 more strongly than KIR2DL1 binds to the human ligand (MHC class I). This interaction mediates inhibitory signalling and suppresses the activation of KIR2DL1-expressing NK cells. We show that KIR2DL1-binding RIFINs are abundant in field-isolated strains from both Africa and Asia and reveal how the two RIFINs bind to KIR2DL1. The RIFIN binding surface of KIR2DL1 is conserved in the cognate activating immune receptor KIR2DS1. We find that KIR2DL1-binding RIFINs can also bind to KIR2DS1, resulting in the activation of KIR2DS1-expressing NK cells. This study demonstrates that activating killer immunoglobulin-like receptors can recruit NK cells to target a pathogen and reveals a potential role for activating immune receptors in controlling malaria infection.
Chromothripsis-associated chromosome 21 amplification orchestrates transformation to blast-phase MPN through targetable overexpression of DYRK1A
Abstract Chromothripsis, the chaotic shattering and repair of chromosomes, is common in cancer. Whether chromothripsis generates actionable therapeutic targets remains an open question. In a cohort of 64 patients in blast phase of a myeloproliferative neoplasm (BP-MPN), we describe recurrent amplification of a region of chromosome 21q (‘chr. 21amp’) in 25%, driven by chromothripsis in a third of these cases. We report that chr. 21amp BP-MPN has a particularly aggressive and treatment-resistant phenotype. DYRK1A, a serine threonine kinase, is the only gene in the 2.7-megabase minimally amplified region that showed both increased expression and chromatin accessibility compared with non-chr. 21amp BP-MPN controls. DYRK1A is a central node at the nexus of multiple cellular functions critical for BP-MPN development and is essential for BP-MPN cell proliferation in vitro and in vivo, and represents a druggable axis. Collectively, these findings define chr. 21amp as a prognostic biomarker in BP-MPN, and link chromothripsis to a therapeutic target.
Translational science at the undergraduate level: awakening talents to overcome the valley of death - case report
Abstract Background: In the biomedical field, translational science is the process of applying basic scientific knowledge to advance clinical research through the creation of new drugs, devices, medical procedures, preventive measures, and diagnostic kits. The Covid-19 pandemic exposed a shortage of professionals trained in translational research, essential for responding to global demands. To drive advancements, researchers must overcome the ‘valley of death’, a critical phase in clinical investigation. In response, CEVAP at São Paulo State University (UNESP), Botucatu, Brazil, has developed a strong 'knowledge industry' centered on Translational Science. As part of its research and innovation efforts, CEVAP has developed two biopharmaceuticals, the fibrin sealant and the apilic antivenom, which are currently in the final stage of development. In 2024, CEVAP began the first Brazilian Contract Development and Manufacturing Organization (CDMO) for developing and producing validated and qualified pilot-scale batches to generate clinical trial material. Case Presentation: The implementation of the optional undergraduate course in Translational Science marks a crucial step in strengthening the ‘knowledge industry’. The program, developed in collaboration with São Paulo’s three public universities (USP, UNESP, and UNICAMP), also involves an international partnership with the University of Oxford’s Department of Pediatrics and the Oxford Research Group LATAM. The successful launch of this course underscores its importance in interdisciplinary education and institutional collaboration. By bridging gaps between research and application, the program equips professionals to meet the growing demand for expertise in translational science. Given the project's success, it will transition into a one-year ‘Qualification in Translational Science’, available to students enrolled in São Paulo state universities. Conclusion: The preparation of these professionals will be strategic for transforming basic research into products for health, saving lives, and combating future pandemics that will emerge around the world.
Clathrin mediates both internalization and vesicular release of triggered T cell receptor at the immunological synapse.
Ligation of T cell receptor (TCR) to peptide-MHC (pMHC) complexes initiates signaling leading to T cell activation and TCR ubiquitination. Ubiquitinated TCR is then either internalized by the T cell or released toward the antigen-presenting cell (APC) in extracellular vesicles. How these distinct fates are orchestrated is unknown. Here, we show that clathrin is first recruited to TCR microclusters by HRS and STAM2 to initiate release of TCR in extracellular vesicles through clathrin- and ESCRT-mediated ectocytosis directly from the plasma membrane. Subsequently, EPN1 recruits clathrin to remaining TCR microclusters to enable trans-endocytosis of pMHC-TCR conjugates from the APC. With these results, we demonstrate how clathrin governs bidirectional membrane exchange at the immunological synapse through two topologically opposite processes coordinated by the sequential recruitment of ecto- and endocytic adaptors. This provides a scaffold for direct two-way communication between T cells and APCs.
Androgens Profile in Blood Serum and Follicular Fluid of Women With Poor Ovarian Response During Controlled Ovarian Stimulation Reveals Differences Amongst POSEIDON Stratification Groups: A Pilot Study.
Patients with poor ovarian response (POR) to exogenous gonadotropins stimulation for assisted reproductive technology (ART) have decreased circulating androgens during spontaneous cycles. The Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) is a 4-tier stratification of women with POR to controlled ovarian stimulation (COH) based on age and biomarkers of ovarian reserve has been proposed to maximize the clinical management of this group for ART. The aim of the present study was to characterize the levels of androgens during COH in follicular fluid (FF) and serum in POSEIDON subgroups and compared them with women of normal ovarian response. Sixty nine consecutive patients undergoing ART were included and testosterone, androstenedione, dehydroepiandrosterone sulfate (DHEA-S), estradiol, sex hormone-binding globulin (SHBG), and insulin-like growth factor 1 (IGF-1) were measured in serum and FF collected at the time of oocyte pick-up. The number of retrieved oocytes was registered for each patient for their allocation to the respective POSEIDON subgroup. The control group comprised 19 women and the POSEIDON group 1 (age < 35, normal ovarian reserve biomarkers) n = 14, group 2 (age ≥ 35, normal ovarian reserve biomarkers) n = 8, group 3 (age < 35, poor ovarian reserve biomarkers) n = 6 and group 4 (age ≥ 35, poor ovarian reserve biomarkers) n = 22. Serum levels of total testosterone, androstenedione and DHEA-S were not different in group 1 vs. control but significantly decreased in group 3 vs. control. DHEA-S in FF was also significantly decreased in group 3 vs. control. In addition, serum testosterone was decreased in groups 2 and 4 vs. control; and serum androstenedione and estradiol were reduced in group 4 vs. control. No differences were observed for estradiol, SHBG and IGF-1 in FF. Finally, a high correlation between serum and FF DHEA-S was observed when data from samples of all groups were pooled. Group 1 did not show hypoandrogenemia however group 3 had low levels of all measured androgens in serum and DHEA-S in FF. Such differences might help to better characterize and/or improve the clinical management of women with POR according to their respective POSEIDON stratification.
Percepciones y creencias sobre criopreservación embrionaria en mujeres y hombres que se realizan técnicas de reproducción asistida en Santiago, Chile.
Background and objetiveAlthough embryo cryopreservation is frequently used as part of assisted reproductive technology, quantitave information addressing how infertile couples live the experience of having cryopreserved embryos is lacking in Chile. The aim of this study is to examine men and women's perception and beliefs regarding their cryopreserved embryos, as well as their perspective on embryo donation and disposition. Methods: 153 women and men with frozen embryos from a public hospital, Instituto de Investigactiones Materno Infantil, and a private clinic, Clínica Las Condes, in Santiago, Chile, responded between May 2015 and May 2016 to an anonymous online survey addressing their perceptions and beliefs concerning their cryopreserved embryos.ResultsRespondents considered their frozen embryos to be equivalent to a child (53.2%) or a potential child (40.7%). Only 8% regard them as an organized group of cells. Over 60% of respondents disagree with destroying surplus embryos or using them for research. Participants from the public hospital are more willing to donate their embryos to another couple than those from the private center (61% vs 40%; P=0.016); 34% of respondents agreed to donate surplus embryos to same sex couples.ConclusionThis study reveals that Chilean couples are emotionally bound to their frozen embryos, and that discarding them is not an option. The results from this survey will help strengthen counseling for couples to enable them to make informed decisions regarding their surplus embryos.
[Prevalence of endometriosis in 287 women undergoing surgical sterilization in Santiago Chile].
BackgroundThe clinical manifestations of endometriosis are infertility, dysmenorrhea, sexuality disturbances, and chronic pelvic pain. It is the cause of 30 to 50% of infertility cases. In developed countries, the prevalence of endometriosis among women undergoing surgical sterilization is approximately 6%.AimTo determine the prevalence of endometriosis among women with proven fertility in Santiago de Chile.Material and methodsReview of surgical protocols of 287 women aged 25 to 49 years, subjected to a surgical sterilization between 2007 and 2011.ResultsEndometriosis was found in 14 of the 287 women (4.9%). In spite of being asymptomatic, five of the 14 women with endometriosis were classified as severe, due to the presence of at least one endometrioma. In order of frequency, the most commonly affected anatomical sites were the ovary, the peritoneum, the posterior cul-de-sac and uterosacral ligaments.ConclusionsOur findings are very similar to those found elsewhere and suggest that fertile women could better tolerate endometriosis than their infertile counterparts.
[Effects of smoking on plasma antimüllerian hormone concentrations among infertile women].
BackgroundSmoking may hamper female fertility, probably modifying ovarian reserve. Antimüllerian hormone (AMH) is an accurate marker for ovarian reserve.AimTo look for an association between smoking status and plasma AMH concentration.Patients and methodsA cohort of 141 infertile women in a university setting in Santiago, Chile was studied. Demographic and smoking data, including the number of cigarettes smoked during the last week, were collected. A blood sample was obtained and kept frozen until determination of AMH by ELISA and follicle stimulating hormone (FSH) and estradiol at day three of the menstrual cycle, by radioimmunoanalysis.ResultsThirty two participants smoked (23%). There were no significant differences in age, parity, body mass index, causes of infertility and day three FSH and estradiol between smokers and nonsmokers. According to a regression analysis, there was a significant decrease in AMH concentration with age and active cigarette smoking. A drop in AMH of -0.189 ng/mL with a unitary change in age and a decrease of -2.29 ng/mL when everything else remains constant, except the smoking status, were established (p < 0.001 and r2 = 0.134). However, no dose response was observed when the number of cigarettes smoked during the last week were introduced in the model. Furthermore, no significant association of plasma AMH with day three plasma FSH and estradiol concentrations was observed.ConclusionsCigarette smoking is associated with decreased AMH plasma concentrations among infertile women. However there was no dose response relationship. The mechanisms underlying this association are unknown and further investigation is required.
Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity
AbstractCancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.
Engineering TCR-controlled fuzzy logic into CAR T cells enhances therapeutic specificity.
Chimeric antigen receptor (CAR) T cell immunotherapy represents a breakthrough in the treatment of hematological malignancies, but poor specificity has limited its applicability to solid tumors. By contrast, natural T cells harboring T cell receptors (TCRs) can discriminate between neoantigen-expressing cancer cells and self-antigen-expressing healthy tissues but have limited potency against tumors. We used a high-throughput platform to systematically evaluate the impact of co-expressing a TCR and CAR on the same CAR T cell. While strong TCR-antigen interactions enhanced CAR activation, weak TCR-antigen interactions actively antagonized their activation. Mathematical modeling captured this TCR-CAR crosstalk in CAR T cells, allowing us to engineer dual TCR/CAR T cells targeting neoantigens (HHATL8F/p53R175H) and human epithelial growth factor receptor 2 (HER2) ligands, respectively. These T cells exhibited superior anti-cancer activity and minimal toxicity against healthy tissue compared with conventional CAR T cells in a humanized solid tumor mouse model. Harnessing pre-existing inhibitory crosstalk between receptors, therefore, paves the way for the design of more precise cancer immunotherapies.
Comparison of different methods for isolating CD8+ T lymphocyte‐derived extracellular vesicles and supramolecular attack particles
AbstractCD8+ T lymphocytes play vital roles in killing infected or deranged host cells, recruiting innate immune cells, and regulating other aspects of immune responses. Like any other cell, CD8+ T cells also produce extracellular particles. These include extracellular vesicles (EVs) and non‐vesicular extracellular particles (NVEPs). T cell‐derived EVs are proposed to mediate cell‐to‐cell signalling, especially in the context of inflammatory responses, autoimmunity, and infectious diseases. CD8+ T cells also produce supramolecular attack particles (SMAPs), which are in the same size range as EVs and mediate a component of T cell mediated killing. The isolation technique selected will have a profound effect on yield, purity, biochemical properties and function of T cell‐derived particles; making it important to directly compare different approaches. In this study, we compared commonly used techniques (membrane spin filtration, ultracentrifugation, or size exclusion liquid chromatography) to isolate particles from activated human CD8+ T cells and validated our results by single‐particle methods, including nanoparticle tracking analysis, flow cytometry, electron microscopy and super‐resolution microscopy of the purified sample as well as bulk proteomics and lipidomics analyses to evaluate the quality and nature of enriched T cell‐derived particles. Our results show that there is a trade‐off between the yield and the quality of T cell‐derived particles. Furthermore, the protein and lipid composition of the particles is dramatically impacted by the isolation technique applied. We conclude that from the techniques evaluated, size exclusion liquid chromatography offers the highest quality of T cell derived EVs and SMAPs with acceptable yields for compositional and functional studies.
Quantifying biomolecular organisation in membranes with brightness-transit statistics.
Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.
Sotrovimab versus usual care in patients admitted to hospital with COVID-19: a randomised, controlled, open-label, platform trial (RECOVERY)
Background: Sotrovimab is a neutralising monoclonal antibody targeting the SARS-COV-2 spike protein that was evaluated in the RECOVERY trial, a randomised, controlled, open-label, platform trial testing treatments for COVID-19. Methods: Patients hospitalised with COVID-19 pneumonia from 107 UK hospitals were randomly allocated to either usual care alone or usual care plus a single 1g infusion of sotrovimab, using web-based unstratified randomisation. Participants were retrospectively categorised as ‘high-antigen’ (the prespecified primary analysis population) if baseline serum SARS-CoV-2 nucleocapsid antigen was above the median concentration, and otherwise as ‘low-antigen’. The primary outcome was 28-day mortality assessed by intention to treat. Recruitment closed on 31 March 2024 when funding ended. ISRCTN (50189673) and clinicaltrials.gov (NCT04381936). Findings: From 4 January 2022 to 19 March 2024, 1723 patients were recruited, 828 allocated sotrovimab and 895 allocated usual care. 720 (42%) were classified as high-antigen, 717 (42%) as low-antigen, and 286 (17%) had unknown antigen status. 1389 (81%) patients were vaccinated, 1179/1438 with known serostatus (82%) had anti-spike antibodies at randomisation, and almost all were infected with Omicron variants. Among high-antigen patients, 82/355 (23%) allocated sotrovimab versus 106/365 (29%) allocated usual care died within 28 days (rate ratio 0.75; 95% CI 0.56-0.99; p=0.046). In an analysis of all randomised patients (regardless of antigen status), 177/828 (21%) allocated sotrovimab versus 201/895 (22%) allocated usual care died within 28 days (rate ratio 0.95; 95% CI 0.77-1.16; p=0.60). Interpretation: In patients hospitalised with COVID-19, sotrovimab was associated with reduced mortality in the primary analysis population who had a high serum SARS-CoV-2 antigen concentration at baseline, but not in the overall population. Treatment options for hospitalised patients are limited, and mortality in those receiving current standard care was high. The emergence of high-level resistance to sotrovimab among subsequent SARS-CoV-2 variants limits its current usefulness, but these results indicate that targeted neutralising antibody therapy could potentially still benefit high-risk hospitalised patients in an era of widespread vaccination and Omicron infection.
Unveiling the structural spectrum of SARS-CoV-2 fusion by in situ cryo-ET.
SARS-CoV-2 entry into host cells is mediated by the spike protein, which drives membrane fusion. While cryo-EM reveals stable prefusion and postfusion conformations of the spike, the transient fusion intermediate states during the fusion process remain poorly understood. Here, we design a near-native viral fusion system that recapitulates SARS-CoV-2 entry and use cryo-electron tomography (cryo-ET) to capture fusion intermediates leading to complete fusion. The spike protein undergoes extensive structural rearrangements, progressing through extended, partially folded, and fully folded intermediates prior to fusion-pore formation, a process that depends on protease cleavage and is inhibited by the WS6 S2 antibody. Upon interaction with ACE2 receptor dimer, spikes cluster at membrane interfaces and following S2' cleavage concurrently transition to postfusion conformations encircling the hemifusion and initial fusion pores in a distinct conical arrangement. S2' cleavage is indispensable for advancing fusion intermediates to the fully folded postfusion state, culminating in membrane integration. Subtomogram averaging reveals that the WS6 S2 antibody binds to the spike's stem-helix, crosslinks and clusters prefusion spikes, as well as inhibits refolding of fusion intermediates. These findings elucidate the entire process of spike-mediated fusion and SARS-CoV-2 entry, highlighting the neutralizing mechanism of S2-targeting antibodies.