Adaptive evolution plays a large role in generating the phenotypic diversity observed in nature, yet current methods are impractical for characterizing the molecular basis and fitness effects of ...large numbers of individual adaptive mutations. Here, we used a DNA barcoding approach to generate the genotype-to-fitness map for adaptation-driving mutations from a Saccharomyces cerevisiae population experimentally evolved by serial transfer under limiting glucose. We isolated and measured the fitness of thousands of independent adaptive clones and sequenced the genomes of hundreds of clones. We found only two major classes of adaptive mutations: self-diploidization and mutations in the nutrient-responsive Ras/PKA and TOR/Sch9 pathways. Our large sample size and precision of measurement allowed us to determine that there are significant differences in fitness between mutations in different genes, between different paralogs, and even between different classes of mutations within the same gene.
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•High-throughput method to survey adaptive mutations in laboratory evolution•Isolation of thousands of independent single adaptive events genome-wide•Bulk fitness measurements and genotyping generates a genotype-to-fitness map•Large sample size allows sensitive detection of fitness differences even within genes
How can the entire suite of adaptive mutations that occur in an evolving population be pinpointed and each of their fitness effects measured?
Abstract
Background
Myocarditis following coronavirus disease 2019 (COVID-19) mRNA vaccines (Pfizer-BioNTech and Moderna) has been increasingly reported. Incidence rates in the general population are ...lacking, with pericarditis rather than myocarditis diagnostic codes being used to estimate background rates. This comparison is critical for balancing the risk of vaccination with the risk of no vaccination.
Methods
A retrospective case series was performed using the Mayo Clinic COVID-19 Vaccine Registry. We measured the incidence rate ratio (IRR) for myocarditis temporally related to COVID-19 mRNA vaccination compared with myocarditis in a comparable population from 2016 through 2020. Clinical characteristics and outcomes of the affected patients were collected. A total of 21 individuals were identified, but ultimately 7 patients met the inclusion criteria for vaccine-associated myocarditis.
Results
The overall IRR of COVID-19–related myocarditis was 4.18 (95% confidence interval CI, 1.63–8.98), which was entirely attributable to an increased IRR among adult males (IRR, 6.69; 95% CI, 2.35–15.52) compared with females (IRR 1.41; 95% CI, .03–8.45). All cases occurred within 2 weeks of a dose of the COVID-19 mRNA vaccine, with the majority occurring within 3 days (range, 1–13) following the second dose (6 of 7 patients, 86%). Overall, cases were mild, and all patients survived.
Conclusions
Myocarditis is a rare adverse event associated with COVID-19 mRNA vaccines. It occurs in adult males with significantly higher incidence than in the background population. Recurrence of myocarditis after a subsequent mRNA vaccine dose is not known at this time.
The incidence rate ratio of myocarditis following coronavirus disease 2019 mRNA vaccination is significantly increased for adult males. Most cases occurred within 3 days (range, 1–13) following the second vaccine dose. Cases were mild, and there were no deaths.
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for hematological malignancies. However, graft-versus-host disease (GVHD) and relapse after allo-HSCT ...remain major impediments to the success of allo-HSCT. Chimeric antigen receptors (CARs) direct tumor cell recognition of adoptively transferred T cells. CD19 is an attractive CAR target, which is expressed in most B cell malignancies, as well as in healthy B cells. Clinical trials using autologous CD19-targeted T cells have shown remarkable promise in various B cell malignancies. However, the use of allogeneic CAR T cells poses a concern in that it may increase risk of the occurrence of GVHD, although this has not been reported in selected patients infused with donor-derived CD19 CAR T cells after allo-HSCT. To understand the mechanism whereby allogeneic CD19 CAR T cells may mediate anti-lymphoma activity without causing a significant increase in the incidence of GVHD, we studied donor-derived CD19 CAR T cells in allo-HSCT and lymphoma models in mice. We demonstrate that alloreactive T cells expressing CD28-costimulated CD19 CARs experience enhanced stimulation, resulting in the progressive loss of both their effector function and proliferative potential, clonal deletion, and significantly decreased occurrence of GVHD. Concurrently, the other CAR T cells that were present in bulk donor T cell populations retained their anti-lymphoma activity in accordance with the requirement that both the T cell receptor (TCR) and CAR be engaged to accelerate T cell exhaustion. In contrast, first-generation and 4-1BB-costimulated CAR T cells increased the occurrence of GVHD. These findings could explain the reduced risk of GVHD occurring with cumulative TCR and CAR signaling.
Persistent smoking may cause adverse outcomes among patients with cancer. Many cancer centers have not fully implemented evidence-based tobacco treatment into routine care.
To determine the ...effectiveness of sustained telephone counseling and medication (intensive treatment) compared with shorter-term telephone counseling and medication advice (standard treatment) to assist patients recently diagnosed with cancer to quit smoking.
This unblinded randomized clinical trial was conducted at Massachusetts General Hospital/Dana-Farber/Harvard Cancer Center and Memorial Sloan Kettering Cancer Center. Adults who had smoked 1 cigarette or more within 30 days, spoke English or Spanish, and had recently diagnosed breast, gastrointestinal, genitourinary, gynecological, head and neck, lung, lymphoma, or melanoma cancers were eligible. Enrollment occurred between November 2013 and July 2017; assessments were completed by the end of February 2018.
Participants randomized to the intensive treatment (n = 153) and the standard treatment (n = 150) received 4 weekly telephone counseling sessions and medication advice. The intensive treatment group also received 4 biweekly and 3 monthly telephone counseling sessions and choice of Food and Drug Administration-approved cessation medication (nicotine replacement therapy, bupropion, or varenicline).
The primary outcome was biochemically confirmed 7-day point prevalence tobacco abstinence at 6-month follow-up. Secondary outcomes were treatment utilization rates.
Among 303 patients who were randomized (mean age, 58.3 years; 170 women 56.1%), 221 (78.1%) completed the trial. Six-month biochemically confirmed quit rates were 34.5% (n = 51 in the intensive treatment group) vs 21.5% (n = 29 in the standard treatment group) (difference, 13.0% 95% CI, 3.0%-23.3%; odds ratio, 1.92 95% CI, 1.13-3.27; P < .02). The median number of counseling sessions completed was 8 (interquartile range, 4-11) in the intensive treatment group. A total of 97 intensive treatment participants (77.0%) vs 68 standard treatment participants (59.1%) reported cessation medication use (difference, 17.9% 95% CI, 6.3%-29.5%; odds ratio, 2.31 95% CI, 1.32-4.04; P = .003). The most common adverse events in the intensive treatment and standard treatment groups, respectively, were nausea (n = 13 and n = 6), rash (n = 4 and n = 1), hiccups (n = 4 and n = 1), mouth irritation (n = 4 and n = 0), difficulty sleeping (n = 3 and n = 2), and vivid dreams (n = 3 and n = 2).
Among smokers recently diagnosed with cancer in 2 National Cancer Institute-designated Comprehensive Cancer Centers, sustained counseling and provision of free cessation medication compared with 4-week counseling and medication advice resulted in higher 6-month biochemically confirmed quit rates. However, the generalizability of the study findings is uncertain and requires further research.
ClinicalTrials.gov Identifier: NCT01871506.
Paradoxical to its importance for generating a diverse T cell repertoire, thymic function progressively declines throughout life. This process has been at least partially attributed to the effects of ...sex steroids, and their removal promotes enhanced thymopoiesis and recovery from immune injury. We show that one mechanism by which sex steroids influence thymopoiesis is through direct inhibition in cortical thymic epithelial cells (cTECs) of Delta-like 4 (Dll4), a Notch ligand crucial for the commitment and differentiation of T cell progenitors in a dose-dependent manner. Consistent with this, sex steroid ablation (SSA) led to increased expression of Dll4 and its downstream targets. Importantly, SSA induced by luteinizing hormone-releasing hormone (LHRH) receptor antagonism bypassed the surge in sex steroids caused by LHRH agonists, the gold standard for clinical ablation of sex steroids, thereby facilitating increased Dll4 expression and more rapid promotion of thymopoiesis. Collectively, these findings not only reveal a novel mechanism underlying improved thymic regeneration upon SSA but also offer an improved clinical strategy for successfully boosting immune function.
A growing number of natural killer (NK) cell-based immunotherapy trials utilize ex vivo expansion to grow and activate allogenic and autologous NK cells prior to administration to patients with ...malignancies. Recent data in both murine and macaque models have shown that adoptively infused ex vivo expanded NK cells have extensive trafficking into liver tissue, with relatively low levels of homing to other sites where tumors often reside, such as the bone marrow or lymph nodes. Here, we evaluated gene and surface expression of molecules involved in cellular chemotaxis in freshly isolated human NK cells compared with NK cells expanded ex vivo using two different feeder cells lines: Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) or K562 cells with membrane-bound (mb) 4-1BB ligand and interleukin (IL)-21. Expanded NK cells had altered expression in a number of genes that encode chemotactic ligands and chemotactic receptors that impact chemoattraction and chemotaxis. Most notably, we observed drastic downregulation of C-X-C chemokine receptor type 4 (CXCR4) and upregulation of C-C chemokine receptor type 5 (CCR5) transcription and phenotypic expression. clustered regularly interspaced short palindromic repeats (CRISPR) gene editing of CCR5 in expanded NK cells reduced cell trafficking into liver tissue and increased NK cell presence in the circulation following infusion into immunodeficient mice. The findings reported here show that ex vivo expansion alters multiple factors that govern NK cell homing and define a novel approach using CRISPR gene editing that reduces sequestration of NK cells by the liver.
To evaluate risk factors for postdischarge sequelae in children and adolescents hospitalized for acute coronavirus disease 2019 (COVID-19) or multisystem inflammatory syndrome in children (MIS-C).
...Multicenter prospective cohort study conducted in 25 United States pediatric hospitals. Patients <21-years-old, hospitalized May 2020 to May 2021 for acute COVID-19 or MIS-C with follow-up 2 to 4 months after admission. We assessed readmissions, persistent symptoms or activity impairment, and new morbidities. Multivariable regression was used to calculate adjusted risk ratios (aRR) and 95% confidence intervals (CI).
Of 358 eligible patients, 2 to 4 month survey data were available for 119 of 155 (76.8%) with acute COVID-19 and 160 of 203 (78.8%) with MIS-C. Thirteen (11%) patients with acute COVID-19 and 12 (8%) with MIS-C had a readmission. Thirty-two (26.9%) patients with acute COVID-19 had persistent symptoms (22.7%) or activity impairment (14.3%) and 48 (30.0%) with MIS-C had persistent symptoms (20.0%) or activity impairment (21.3%). For patients with acute COVID-19, persistent symptoms (aRR, 1.29 95% CI, 1.04-1.59) and activity impairment (aRR, 1.37 95% CI, 1.06-1.78) were associated with more organ systems involved. Patients with MIS-C and pre-existing respiratory conditions more frequently had persistent symptoms (aRR, 3.09 95% CI, 1.55-6.14) and those with obesity more frequently had activity impairment (aRR, 2.52 95% CI, 1.35-4.69). New morbidities were infrequent (9% COVID-19, 1% MIS-C).
Over 1 in 4 children hospitalized with acute COVID-19 or MIS-C experienced persistent symptoms or activity impairment for at least 2 months. Patients with MIS-C and respiratory conditions or obesity are at higher risk of prolonged recovery.
This article aims to describe the clinical manifestations and management of COVID-19 in patients with primary and secondary B cell deficient states. We describe the epidemiologic and clinical ...features as well as unique management paradigm including isolation precautions with COVID-19. We then focus upon primary and secondary preventive approaches including vaccination and pre- as well as post-exposure prophylaxis. Further, we elaborate upon the important disease specific risk factors in these patients and the need to conduct prospective clinical trials to develop individualized management strategies in this population.
BackgroundAdoptive transfer of natural killer (NK) cells with augmented antibody-dependent cellular cytotoxicity (ADCC) capabilities and resistance to CD38 targeting has the potential to enhance the ...clinical anti-myeloma activity of daratumumab (DARA). Therefore, we sought to develop an efficient CRISPR/Cas9-based gene editing platform to disrupt CD38 expression (CD38 knockout (KO)) in ex vivo expanded NK cells and simultaneously arm CD38KO NK cells with a high-affinity CD16 (CD16-158V) receptor.MethodsCD38KO human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38KO NK cells and targeted gene insertion at the CD38 locus to mediate gene knockin (KI). The capacity of these gene-edited NK cells to persist and mediate ADCC in the presence of DARA was tested in vitro and in a MM.1S xenograft mouse model.ResultsHighly efficient CD38 gene disruption was achieved in ex vivo expanded NK cells without affecting their proliferative or functional capacity. CD38 KO conferred resistance to DARA-induced NK cell fratricide, enabling persistence and augmented ADCC against myeloma cell lines in the presence of DARA in vitro and in a MM.1S xenograft mouse model. CD38KO NK cells could be further modified by transfection with mRNA encoding a CD16-158V receptor, resulting in augmented DARA-mediated ADCC. Finally, we observed that a homology-directed repair template targeted to the CD38 locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38KO/CD16KI NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo.ConclusionsAdoptive immunotherapy using ex vivo expanded CD38KO/CD16KI NK cells has the potential to boost the clinical efficacy of DARA. By incorporating complementary genetic engineering strategies into a CD38 KO manufacturing platform, we generated NK cells with substantially augmented CD38-directed antitumor activity, establishing a strong rationale for exploring this immunotherapy strategy in the clinic.
The ability of natural killer (NK) cells to mediate antitumor effects following adoptive transfer is dependent on their capacity to traffic to the microenvironment where tumors reside. Recent studies ...have shown that cytokine-activated and ex vivo-expanded NK cells lack or express at low levels homing receptors required to achieve tissue-specific tumor targeting by cells administered intravenously. In this chapter, we describe a method to enhance NK cell homing toward specific chemoattractants expressed in secondary lymphoid tissues through genetic modification of NK cells using mRNA electroporation. The method described here is scalable, cGMP-compliant, and offers a strategy to bolster the efficacy of adoptive NK cell immunotherapy for the treatment of hematological malignancies in the clinic.
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