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During antiretroviral therapy, CD4 lymphocyte count increases are modest in some patients despite virologic control. We explored whether polymorphisms in genes important for T cell expansion, survival, and apoptosis are associated with the magnitude of CD4 lymphocyte count recovery during antiretroviral therapy. We studied treatment-naive individuals who achieved sustained control of plasma viremia (<400 HIV-1 RNA copies/mL) for at least 48 weeks after initiation of antiretroviral therapy and compared genotypes among individuals who had an increase of either <200 or >/=200 CD4 cells/mm(3) from baseline. A total of 137 single-nucleotide polymorphisms across 17 genes were characterized in 873 study participants. In multivariate analyses that controlled for clinical variables, polymorphisms in genes encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF-alpha , Bcl-2-interacting molecule (Bim), interleukin (IL)-15, and IL-15 receptor alpha chain (IL-15R alpha ) were associated with the magnitude of the increase in CD4 lymphocyte count, as were haplotypes in genes encoding interferon-alpha, IL-2, and IL-15R alpha (P<.05, for each). Multifactor dimensionality reduction identified a gene-gene interaction between IL-2/IL-15 receptor common beta chain and IL-2/IL-7/IL-15 receptor common gamma chain. Immune recovery during antiretroviral therapy is a complex phenotype that is influenced by multiple genetic variants. Future studies should validate these tentative associations and define underlying mechanisms. Copyright © 2006 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Disruptions in microbial metabolite interactions due to gut microbiome dysbiosis and metabolomic shifts may contribute to Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and other immune-related conditions. The aryl hydrocarbon receptor (AhR), activated upon binding various tryptophan metabolites, modulates host immune responses. This study investigates whether the metabolic diversity-the concentration distribution-of bacterial indole pathway metabolites can differentiate bacterial strains and classify ME/CFS samples. A fast targeted liquid chromatography-parallel reaction monitoring method at a rate of 4 minutes per sample was developed for large-scale analysis. This method revealed significant metabolic differences in indole derivatives among B. uniformis strains cultured from human isolates. Principal component analysis identified two major components (PC1, 68.9%; PC2, 18.7%), accounting for 87.6% of the variance and distinguishing two distinct B. uniformis clusters. The metabolic difference between clusters was particularly evident in the relative contributions of indole-3-acrylate and indole-3-aldehyde. We further measured concentration distributions of indole derivatives in ME/CFS by analyzing fecal samples from 10 patients and 10 healthy controls using the fast targeted metabolomics method. An AdaBoost-LOOCV model achieved moderate classification success with a mean LOOCV accuracy of 0.65 (Control: precision of 0.67, recall of 0.60, F1-score of 0.63; ME/CFS: precision of 0.64, recall of 0.7000, F1-score of 0.67). These results suggest that the metabolic diversity of indole derivatives from tryptophan degradation, facilitated by the fast targeted metabolomics and machine learning, is a potential biomarker for differentiating bacterial strains and classifying ME/CFS samples. Mass spectrometry datasets are accessible at the National Metabolomics Data Repository (ST002308, DOI: 10.21228/M8G13Q; ST003344, DOI: 10.21228/M8RJ9N; ST003346, DOI: 10.21228/M8RJ9N).

Chronic diseases like ME/CFS and long COVID exhibit high heterogeneity with multifactorial etiology and progression, complicating diagnosis and treatment. To address this, we developed BioMapAI, an explainable Deep Learning framework using the richest longitudinal multi-'omics dataset for ME/CFS to date. This dataset includes gut metagenomics, plasma metabolome, immune profiling, blood labs, and clinical symptoms. By connecting multi-'omics to asymptom matrix, BioMapAI identified both disease- and symptom-specific biomarkers, reconstructed symptoms, and achieved state-of-the-art precision in disease classification. We also created the first connectivity map of these 'omics in both healthy and disease states and revealed how microbiome-immune-metabolome crosstalk shifted from healthy to ME/CFS. Thus, we proposed several innovative mechanistic hypotheses for ME/CFS: Disrupted microbial functions - SCFA (butyrate), BCAA (amino acid), tryptophan, benzoate - lost connection with plasma lipids and bile acids, and activated inflammatory and mucosal immune cells (MAIT, γδT cells) with INFγ and GzA secretion. These abnormal dynamics are linked to key disease symptoms, including gastrointestinal issues, fatigue, and sleep problems., Competing Interests: Competing Interests Dr. Suzanne D. Vernon is affiliated and has a financial interest with The BioCollective, a company that provided the BioCollector, the collection kit used for at home stool collection discussed in this manuscript. No other authors have competing interests.

Mucosal-associated invariant T (MAIT) cells play diverse roles in cancer, infectious diseases, and immunotherapy. This review explores their intricate involvement in cancer, from early detection to their dual functions in promoting inflammation and mediating anti-tumor responses. Within the solid tumor microenvironment (TME), MAIT cells can acquire an 'exhausted' state and secrete tumor-promoting cytokines. On the other hand, MAIT cells are highly cytotoxic, and there is evidence that they may have an anti-tumor immune response. The frequency of MAIT cells and their subsets has also been shown to have prognostic value in several cancer types. Recent innovative approaches, such as programming MAIT cells with chimeric antigen receptors (CARs), provide a novel and exciting approach to utilizing these cells in cell-based cancer immunotherapy. Because MAIT cells have a restricted T cell receptor (TCR) and recognize a common antigen, this also mitigates potential graft-versus-host disease (GVHD) and opens the possibility of using allogeneic MAIT cells as off-the-shelf cell therapies in cancer. Additionally, we outline the interactions of MAIT cells with the microbiome and their critical role in infectious diseases and how this may impact the tumor responses of these cells. Understanding these complex roles can lead to novel therapeutic strategies harnessing the targeting capabilities of MAIT cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yigit, Basoglu and Unutmaz.)

Aryl hydrocarbon receptor (AhR) is a transcription factor that regulates gene expression upon ligand activation, enabling microbiota-dependent induction, training, and function of the host immune system. A spectrum of metabolites, encompassing indole and tryptophan derivatives, have been recognized as activators. This work introduces an integrated, mass spectrometry-centric workflow that employs a bioassay-guided, fractionation-based methodology for the identification of AhR activators derived from human bacterial isolates. By leveraging the workflow efficiency, the complexities inherent in metabolomics profiling are significantly reduced, paving the way for an in-depth and focused mass spectrometry analysis of bioactive fractions isolated from bacterial culture supernatants. Validation of AhR activator candidates used multiple criteria─MS/MS of the synthetic reference compound, bioassay of AhR activity, and elution time confirmation using a C-13 isotopic reference─and was demonstrated for N -formylkynurenine (NFK). The workflow reported provides a roadmap update for improved efficiency of identifying bioactive metabolites using mass spectrometry-based metabolomics. Mass spectrometry datasets are accessible at the National Metabolomics Data Repository (PR001479, Project DOI: 10.21228/M8JM7Q).

Background & Aims: Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-β docking molecule. While the immune regulatory properties of GARP on blood cells have been studied, the function of GARP on tissue stromal cells remains unclear. Here, we investigate the role of GARP expressed on hepatic stellate cells (HSCs) in the development of liver fibrosis., Methods: The function of GARP on HSCs was explored in toxin-induced and metabolic liver fibrosis models, using conditional GARP-deficient mice or a newly generated inducible system for HSC-specific gene ablation. Primary mouse and human HSCs were isolated to evaluate the contribution of GARP to the activation of latent TGF-β. Moreover, cell contraction of HSCs in the context of TGF-β activation was tested in a GARP-dependent fashion., Results: Mice lacking GARP in HSCs were protected from developing liver fibrosis. Therapeutically deleting GARP on HSCs alleviated the fibrotic process in established disease. Furthermore, natural killer T cells exacerbated hepatic fibrosis by inducing GARP expression on HSCs through IL-4 production. Mechanistically, GARP facilitated fibrogenesis by activating TGF-β and enhancing endothelin-1-mediated HSC contraction. Functional GARP was expressed on human HSCs and significantly upregulated in the livers of patients with fibrosis. Lastly, deletion of GARP on HSCs did not augment inflammation or liver damage., Conclusions: GARP expressed on HSCs drives the development of liver fibrosis via cell contraction-mediated activation of latent TGF-β. Considering that systemic blockade of TGF-β has major side effects, we highlight a therapeutic niche provided by GARP and surface-mediated TGF-β activation. Thus, our findings suggest an important role of GARP on HSCs as a promising target for the treatment of liver fibrosis., Impact and Implications: Liver fibrosis represents a substantial and increasing public health burden globally, for which specific treatments are not available. Glycoprotein A repetitions predominant (GARP) is a membrane protein that functions as a latent TGF-β docking molecule. Here, we show that GARP expressed on hepatic stellate cells drives the development of liver fibrosis. Our findings suggest GARP as a novel target for the treatment of fibrotic disease., (Copyright © 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Introduction: Cognitive impairment is experienced by people with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and post-acute sequelae of COVID-19 (PASC). Patients report difficulty remembering, concentrating, and making decisions. Our objective was to determine whether orthostatic hemodynamic changes were causally linked to cognitive impairment in these diseases., Methods: This prospective, observational cohort study enrolled PASC, ME/CFS, and healthy controls. All participants underwent clinical evaluation and assessment that included brief cognitive testing before and after an orthostatic challenge. Cognitive testing measured cognitive efficiency which is defined as the speed and accuracy of subject's total correct responses per minute. General linear mixed models were used to analyze hemodynamics and cognitive efficiency during the orthostatic challenge. Additionally, mediation analysis was used to determine if hemodynamic instability induced during the orthostatic challenge mediated the relationship between disease status and cognitive impairment., Results: Of the 276 participants enrolled, 256 were included in this study (34 PASC, 71 < 4 year duration ME/CFS, 69 > 10 year ME/CFS duration, and 82 healthy controls). Compared to healthy controls, the disease cohorts had significantly lower cognitive efficiency scores immediately following the orthostatic challenge. Cognitive efficiency remained low for the >10 year ME/CFS 2 and 7 days after orthostatic challenge. Narrow pulse pressure less than 25% of systolic pressure occurred at 4 and 5 min into the orthostatic challenge for the PASC and ME/CFS cohorts, respectively. Abnormally narrow pulse pressure was associated with slowed information processing in PASC patients compared to healthy controls (-1.5, p  = 0.04). Furthermore, increased heart rate during the orthostatic challenge was associated with a decreased procedural reaction time in PASC and < 4 year ME/CFS patients who were 40 to 65 years of age., Discussion: For PASC patients, both their disease state and hemodynamic changes during orthostatic challenge were associated with slower reaction time and decreased response accuracy during cognitive testing. Reduced cognitive efficiency in <4 year ME/CFS patients was associated with higher heart rate in response to orthostatic stress. Hemodynamic changes did not correlate with cognitive impairment for >10 year ME/CFS patients, but cognitive impairment remained. These findings underscore the need for early diagnosis to mitigate direct hemodynamic and other physiological effects on symptoms of cognitive impairment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Day, Yellman, Hammer, Rond, Bell, Abbaszadeh, Stoddard, Unutmaz, Bateman and Vernon.)

The genome is pervasively transcribed to produce a vast array of non-coding RNAs (ncRNAs). Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides and are best known for their ability to regulate gene expression. Enhancer RNAs (eRNAs) are subclass of lncRNAs that are synthesized from enhancer regions and have also been shown to coordinate gene expression. The biological function and significance of most lncRNAs and eRNAs remain to be determined. Epithelial to mesenchymal transition (EMT) is a ubiquitous cellular process that occurs during cellular migration, homeostasis, fibrosis, and cancer-cell metastasis. EMT-transcription factors, such as SNAI1 induce a complex transcriptional program that coordinates the morphological and molecular changes associated with EMT. Such complex transcriptional programs are often subject to coordination by networks of ncRNAs and thus can be leveraged to identify novel functional ncRNA loci. Here, using a genome-wide CRISPR activation (CRISPRa) screen targeting ∼10,000 lncRNA loci we identified ncRNA loci that could either promote or attenuate EMT. We discovered a novel locus that we named SCREEM ( SNAI1 cis-regulatory eRNAs expressed in monocytes). The SCREEM locus contained a cluster of eRNAs that when activated using CRISPRa induced expression of the neighboring gene SNAI1 , driving concomitant EMT. However, the SCREEM eRNA transcripts themselves appeared dispensable for the induction of SNAI1 expression. Interestingly, the SCREEM eRNAs and SNAI1 were co-expressed in activated monocytes, where the SCREEM locus demarcated a monocyte-specific super-enhancer. These findings suggest a potential role for SNAI1 in monocytes. Exploration of the SCREEM-SNAI axis could reveal novel aspects of monocyte biology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Uthaya Kumar, Yurieva, Grassmann, Kozhaya, McBride, Unutmaz and Williams.)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, debilitating disorder manifesting as severe fatigue and post-exertional malaise. The etiology of ME/CFS remains elusive. Here, we present a deep metagenomic analysis of stool combined with plasma metabolomics and clinical phenotyping of two ME/CFS cohorts with short-term (<4 years, n = 75) or long-term disease (>10 years, n = 79) compared with healthy controls (n = 79). First, we describe microbial and metabolomic dysbiosis in ME/CFS patients. Short-term patients showed significant microbial dysbiosis, while long-term patients had largely resolved microbial dysbiosis but had metabolic and clinical aberrations. Second, we identified phenotypic, microbial, and metabolic biomarkers specific to patient cohorts. These revealed potential functional mechanisms underlying disease onset and duration, including reduced microbial butyrate biosynthesis and a reduction in plasma butyrate, bile acids, and benzoate. In addition to the insights derived, our data represent an important resource to facilitate mechanistic hypotheses of host-microbiome interactions in ME/CFS., Competing Interests: Declaration of interests S.D.V. is affiliated and has a financial interest with The BioCollective, a company that provided the BioCollector, the collection kit used for at home stool collection discussed in this manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Importance: Early and accurate diagnosis and treatment of Long COVID, clinically known as post-acute sequelae of COVID-19 (PASC), may mitigate progression to chronic diseases such as myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Our objective was to determine the utility of the DePaul Symptom Questionnaire (DSQ) to assess the frequency and severity of common symptoms of ME/CFS, to diagnose and monitor symptoms in patients with PASC., Methods: This prospective, observational cohort study enrolled 185 people that included 34 patients with PASC that had positive COVID-19 test and persistent symptoms of >3 months and 151 patients diagnosed with ME/CFS. PASC patients were followed over 1 year and responded to the DSQ at baseline and 12 months. ME/CFS patients responded to the DSQ at baseline and 1 year later. Changes in symptoms over time were analyzed using a fixed-effects model to compute difference-in-differences estimates between baseline and 1-year follow-up assessments., Participants: Patients were defined as having PASC if they had a previous positive COVID-19 test, were experiencing symptoms of fatigue, post-exertional malaise, or other unwellness for at least 3 months, were not hospitalized for COVID-19, had no documented major medical or psychiatric diseases prior to COVID-19, and had no other active and untreated disease processes that could explain their symptoms. PASC patients were recruited in 2021. ME/CFS patients were recruited in 2017., Results: At baseline, patients with PASC had similar symptom severity and frequency as patients with ME/CFS and satisfied ME/CFS diagnostic criteria. ME/CFS patients experienced significantly more severe unrefreshing sleep and flu-like symptoms. Five symptoms improved significantly over the course of 1 year for PASC patients including fatigue, post-exertional malaise, brain fog, irritable bowel symptoms and feeling unsteady. In contrast, there were no significant symptom improvements for ME/CFS patients., Conclusion and Relevance: There were considerable similarities between patients with PASC and ME/CFS at baseline. However, symptoms improved for PASC patients over the course of a year but not for ME/CFS patients. PASC patients with significant symptom improvement no longer met ME/CFS clinical diagnostic criteria. These findings indicate that the DSQ can be used to reliably assess and monitor PASC symptoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Oliveira, Jason, Unutmaz, Bateman and Vernon.)

Background: Long COVID describes a condition with symptoms that linger for months to years following acute COVID-19. Many of these Long COVID symptoms are like those experienced by patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)., Objective: We wanted to determine if people with Long COVID experienced post-exertional malaise (PEM), the hallmark symptom of ME/CFS, and if so, how it compared to PEM experienced by patients with ME/CFS., Methods: A questionnaire that asked about the domains of PEM including triggers, experience, recovery, and prevention was administered to 80 people seeking care for Long COVID at Bateman Horne Center. Their responses were compared to responses about PEM given by 151 patients with ME/CFS using chi-square tests of independence., Results: All but one Long COVID respondent reported having PEM. There were many significant differences in the types of PEM triggers, symptoms experienced during PEM, and ways to recover and prevent PEM between Long COVID and ME/CFS. Similarities between Long COVID and ME/CFS included low and medium physical and cognitive exertion to trigger PEM, symptoms of fatigue, pain, immune reaction, neurologic, orthostatic intolerance, and gastrointestinal symptoms during PEM, rest to recover from PEM, and pacing to prevent PEM., Conclusion: People with Long COVID experience PEM. There were significant differences in PEM experienced by people with Long COVID compared to patients with ME/CFS. This may be due to the newness of Long COVID, not knowing what exertional intolerance is or how to manage it.

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic-flow based 3D bioprinted multi-scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre-defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose-dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8 + T cells for 24 or 72 h. Extensive CAR-T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically-relevant tumor model for future translation of anti-cancer therapies to personalized medicine., Competing Interests: Conflict of Interest I.T.O. has an equity stake in Biolife4D and is a member of the scientific advisory board for Biolife4D and Brinter. Other authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Objectives: Despite advances in antibody treatments and vaccines, COVID-19 caused by SARS-CoV-2 infection remains a major health problem resulting in excessive morbidity and mortality and the emergence of new variants has reduced the effectiveness of current vaccines., Methods: Here, as a proof-of-concept, we engineered primary CD8 T cells to express SARS-CoV-2 Spike protein-specific CARs, using the extracellular region of ACE2 and demonstrated their highly specific and potent cytotoxicity towards Spike-expressing target cells. To improve on this concept as a potential therapeutic, we developed a bispecific T cell engager combining ACE2 with an anti-CD3 scFv (ACE2-Bite) to target infected cells and the virus., Results: As in CAR-T cell approach, ACE2-Bite endowed cytotoxic cells to selectively kill Spike-expressing targets. Furthermore, ACE2-Bite neutralized the pseudoviruses of SARS-CoV, SARS-CoV-2 wild-type, and variants including Delta and Omicron, as a decoy protein. Remarkably, ACE2-Bite molecule showed a higher binding and neutralization affinity to Delta and Omicron variants compared to SARS-CoV-2 wild-type Spike proteins., Conclusion: In conclusion, these results suggest the potential of this approach as a variant-proof, therapeutic strategy for future SARS-CoV-2 variants, employing both humoral and cellular arms of the adaptive immune response., Competing Interests: MD and DU are inventors in a provisional patent application., (© 2022 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Engineering immune cells with chimeric Ag receptors (CARs) is a promising technology in cancer immunotherapy. Besides classical cytotoxic CD8 + T cells, innate cell types such as NK cells have also been used to generate CAR-T or CAR-NK cells. In this study, we devised an approach to program a nonclassical cytotoxic T cell subset called mucosal-associated invariant T (MAIT) cells into effective CAR-T cells against B cell lymphoma and breast cancer cells. Accordingly, we expressed anti-CD19 and anti-Her2 CARs in activated primary human MAIT cells and CD8 + T cells, expanded them in vitro, and compared their cytotoxicity against tumor cell targets. We show upon activation through CARs that CAR-MAIT cells exhibit high levels of cytotoxicity toward target cells, comparable to CD8 + CAR-T cells, but interestingly expressed lower levels of IFN-γ than conventional CAR CD8 + T cells. Additionally, in the presence of vitamin B 2 metabolite 5-ARU (5-amino-4-d-ribitylaminouracil dihydrochloride), which is a conserved compound that activates MAIT cells through MHC class I-related (MR1) protein, MAIT cells killed MR1-expressing target breast cancer and B cell lymphoma cell lines in a dose-dependent manner. Thus, MAIT cells can be genetically edited as CAR-T cells or mobilized and expanded by MR1 ligands as an off-the-shelf novel approach to cell-based cancer immunotherapy strategies while being comparable to conventional methods in effectivity., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8 + T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies., (© 2022 IOP Publishing Ltd.)

Aims: Studies have linked severe hyperoxia, or prolonged exposure to very high oxygen levels, with worse clinical outcomes. This study investigated the role of epidermal growth factor receptor (EGFR) in hyperoxia-induced lung injury at very high oxygen levels (>95%)., Results: Effects of severe hyperoxia (100% oxygen) were studied in mice with genetically inhibited EGFR and wild-type littermates. Despite the established role of EGFR in lung repair, EGFR inhibition led to improved survival and reduced acute lung injury, which prompted an investigation into this protective mechanism. Endothelial EGFR genetic knockout did not confer protection. EGFR inhibition led to decreased levels of cleaved caspase-3 and poly (ADP-ribosyl) polymerase (PARP) and decreased terminal dUTP nick end labeling- (TUNEL-) positive staining in alveolar epithelial cells and reduced ERK activation, which suggested reduced apoptosis in vivo . EGFR inhibition decreased hyperoxia (95%)-induced apoptosis and ERK in murine alveolar epithelial cells in vitro , and CRISPR-mediated EGFR deletion reduced hyperoxia-induced apoptosis and ERK in human alveolar epithelial cells in vitro . Innovation . This work defines a protective role of EGFR inhibition to decrease apoptosis in lung injury induced by 100% oxygen. This further characterizes the complex role of EGFR in acute lung injury and outlines a novel hyperoxia-induced cell death pathway that warrants further study., Conclusion: In conditions of severe hyperoxia (>95% for >24 h), EGFR inhibition led to improved survival, decreased lung injury, and reduced cell death. These findings further elucidate the complex role of EGFR in acute lung injury., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2022 Zachary M. Harris et al.)

The determinants of severe COVID-19 in healthy adults are poorly understood, which limits the opportunity for early intervention. We present a multiomic analysis using machine learning to characterize the genomic basis of COVID-19 severity. We use single-cell multiome profiling of human lungs to link genetic signals to cell-type-specific functions. We discover >1,000 risk genes across 19 cell types, which account for 77% of the SNP-based heritability for severe disease. Genetic risk is particularly focused within natural killer (NK) cells and T cells, placing the dysfunction of these cells upstream of severe disease. Mendelian randomization and single-cell profiling of human NK cells support the role of NK cells and further localize genetic risk to CD56 bright NK cells, which are key cytokine producers during the innate immune response. Rare variant analysis confirms the enrichment of severe-disease-associated genetic variation within NK-cell risk genes. Our study provides insights into the pathogenesis of severe COVID-19 with potential therapeutic targets., Competing Interests: Declaration of interests M.P.S. is a co-founder and member of the scientific advisory board of Personalis, Qbio, January, SensOmics, Protos, Mirvie, NiMo, Onza, and Oralome. He is also on the scientific advisory board of Danaher, Genapsys, and Jupiter. No other authors have competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Characterization of neutralization antibodies to SARS-CoV-2 infection or vaccination in children and young adults with inflammatory bowel disease (IBD) receiving biologic therapies is crucial., Methods: We performed a prospective longitudinal cohort study evaluating SARS-CoV-2 spike protein receptor binding domain (S-RBD) IgG positivity along with consistent clinical symptoms in patients with IBD receiving infliximab or vedolizumab. Serum was also obtained following immunization with approved vaccines. The IgG antibody to the spike protein binding domain of SARS-CoV-2 was assayed with a fluorescent bead-based immunoassay that takes advantage of the high dynamic range of fluorescent molecules using flow cytometry. A sensitive and high-throughput neutralization assay that incorporates SARS-CoV-2 spike protein onto a lentivirus and measures pseudoviral entry into ACE2-angiotensin converting enzyme 2 (ACE2) expressing human embryonic kidney 293 (HEK-293) cells was used., Results: There were 436 patients enrolled (mean age, 17 years, range 2-26 years; 58% male; 71% Crohn's disease, 29% ulcerative colitis, IBD-unspecified). Forty-four (10%) of enrolled subjects had SARS-CoV-2 S-RBD IgG antibodies. Compared to non-IBD adults (ambulatory) and hospitalized pediatric patients with PCR documented SARS-CoV-2 infection, S-RBD IgG antibody levels were significantly lower in the IBD cohort and by 6 months post infection most patients lacked neutralizing antibody. Following vaccination (n = 33), patients had a 15-fold higher S-RBD antibody response in comparison with natural infection, and all developed neutralizing antibodies to both wild type and variant SARS-CoV-2., Conclusions: The lower and less durable SARS-CoV-2 S-RBD IgG response to natural infection in IBD patients receiving biologics puts them at risk of reinfection. The robust response to immunization is likely protective., (© 2021 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.)

Background: PD-1 marks exhausted T cells, with weak effector functions. Adults living with human immunodeficiency virus (HIV) have increased levels of PD-1+ CD8 T cells that correlate with HIV disease progression, yet little is known about the role of PD-1+ CD8 T cells in children with perinatal HIV., Methods: We enrolled 76 Kenyan children with perinatal HIV and 43 children who were HIV unexposed and quantified PD-1 levels on CD8 T cells; their coexpression with immune checkpoints (ICs) 2B4, CD160, and TIM3; correlates with immune activation and HIV disease progression; and HIV-specific and -nonspecific proliferative responses., Results: PD-1+ CD8 T-cell frequencies are elevated in children with perinatal HIV and associated with disease progression. The majority of PD-1+ CD8 T cells coexpress additional ICs. ART initiation lowers total PD-1 levels and coexpression of multiple ICs. The frequency of PD-1+2B4+CD160+TIM3- in PD-1+ CD8 T cells predicts weaker HIV-specific proliferative responses, suggesting that this subset is functionally exhausted., Conclusions: Children with perinatal HIV have high levels of PD-1+ CD8 T cells that are a heterogeneous population differentially coexpressing multiple ICs. Understanding the complex interplay of ICs is essential to guide the development of PD-1-directed immunotherapies for pediatric HIV remission and cure., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Over the last few decades, the world has witnessed multiple viral pandemics, the current severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) pandemic being the worst and most devastating one, claiming millions of lives worldwide. Physicians, scientists, and engineers worldwide have joined hands in dealing with the current situation at an impressive speed and efficiency. One of the major reasons for the delay in response is our limited understanding of the mechanism of action and individual effects of the virus on different tissues and organs. Advances in 3D bioprinting have opened up a whole new area to explore and utilize the technology in fabricating models of these tissues and organs, recapitulating in vivo environment. These biomimetic models can not only be utilized in learning the infection pathways and drug toxicology studies but also minimize the need for animal models and shorten the time span for human clinical trials. The current review aims to integrate the existing developments in bioprinting techniques, and their implementation to develop tissue models, which has implications for SARS-CoV-2 infection. Future translation of these models has also been discussed with respect to the pandemic., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Metastatic breast cancer is one of the deadliest forms of malignancy, primarily driven by its characteristic micro-environment comprising cancer cells interacting with stromal components. These interactions induce genetic and metabolic alterations creating a conducive environment for tumor growth. In this study, a physiologically relevant 3D vascularized breast cancer micro-environment is developed comprising of metastatic MDA-MB-231 cells and human umbilical vein endothelial cells loaded in human dermal fibroblasts laden fibrin, representing the tumor stroma. The matrix, as well as stromal cell density, impacts the transcriptional profile of genes involved in tumor angiogenesis and cancer invasion, which are hallmarks of cancer. Cancer-specific canonical pathways and activated upstream regulators are also identified by the differential gene expression signatures of these composite cultures. Additionally, a tumor-associated vascular bed of capillaries is established exhibiting dilated vessel diameters, representative of in vivo tumor physiology. Further, employing aspiration-assisted bioprinting, cancer-endothelial crosstalk, in the form of collective angiogenesis of tumor spheroids bioprinted at close proximity, is identified. Overall, this bottom-up approach of tumor micro-environment fabrication provides an insight into the potential of in vitro tumor models and enables the identification of novel therapeutic targets as a preclinical drug screening platform., (© 2021 Wiley-VCH GmbH.)

Aging has emerged as the greatest and most prevalent risk factor for the development of severe COVID-19 infection and death following exposure to the SARS-CoV-2 virus. The presence of multiple co-existing chronic diseases and conditions of aging further enhances this risk. Biological aging not only enhances the risk of chronic diseases, but the presence of such conditions further accelerates varied biological processes or "hallmarks" implicated in aging. Given growing evidence that it is possible to slow the rate of many biological aging processes using pharmacological compounds has led to the proposal that such geroscience-guided interventions may help enhance immune resilience and improve outcomes in the face of SARS-CoV-2 infection. Our review of the literature indicates that most, if not all, hallmarks of aging may contribute to the enhanced COVID-19 vulnerability seen in frail older adults. Moreover, varied biological mechanisms implicated in aging do not function in isolation from each other, and exhibit intricate effects on each other. With all of these considerations in mind, we highlight limitations of current strategies mostly focused on individual single mechanisms, and we propose an approach which is far more multidisciplinary and systems-based emphasizing network topology of biological aging and geroscience-guided approaches to COVID-19.

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.