A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGE hi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS. </p

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.</p

A myeloid program associated with COVID-19 severity is decreased by therapeutic blockade of IL-6 signaling

Altered myeloid inflammation and lymphopenia are hallmarks of severe infections. We identified the upregulated EN-RAGE gene program in airway and blood myeloid cells from patients with acute lung injury from SARS-CoV-2 or other causes across 7 cohorts. This program was associated with greater clinical severity and predicted future mechanical ventilation and death. EN-RAGEhi myeloid cells express features consistent with suppressor cell functionality, including low HLA-DR and high PD-L1. Sustained EN-RAGE program expression in airway and blood myeloid cells correlated with clinical severity and increasing expression of T cell dysfunction markers. IL-6 upregulated many EN-RAGE program genes in monocytes in vitro. IL-6 signaling blockade by tocilizumab in a placebo-controlled clinical trial led to rapid normalization of EN-RAGE and T cell gene expression. This identifies IL-6 as a key driver of myeloid dysregulation associated with worse clinical outcomes in COVID-19 patients and provides insights into shared pathophysiological mechanisms in non-COVID-19 ARDS.</p

Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19

Neutralizing autoantibodies against type I interferons (IFNs) have been found in some patients with critical coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the prevalence of these antibodies, their longitudinal dynamics across the disease severity scale, and their functional effects on circulating leukocytes remain unknown. Here, in 284 patients with COVID-19, we found type I IFN–specific autoantibodies in peripheral blood samples from 19% of patients with critical disease and 6% of patients with severe disease. We found no type I IFN autoantibodies in individuals with moderate disease. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 patients with COVID-19 and 26 non–COVID-19 controls revealed a lack of type I IFN–stimulated gene (ISG-I) responses in myeloid cells from patients with critical disease. This was especially evident in dendritic cell populations isolated from patients with critical disease producing type I IFN–specific autoantibodies. Moreover, we found elevated expression of the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) on the surface of monocytes isolated from patients with critical disease early in the disease course. LAIR1 expression is inversely correlated with ISG-I expression response in patients with COVID-19 but is not expressed in healthy controls. The deficient ISG-I response observed in patients with critical COVID-19 with and without type I IFN–specific autoantibodies supports a unifying model for disease pathogenesis involving ISG-I suppression through convergent mechanisms

Investigating Mechanisms of Robustness in BRCA -Mutated Breast and Ovarian Cancers

The BRCA1 and BRCA2 (BRCA) genes are two tumor suppressors that when mutated, predispose patients to breast and ovarian cancer. The BRCA genes encode proteins that mediate the repair of DNA double strand breaks. Functional loss of the BRCA genes is detrimental to the integrity of the genome because without access to functional BRCA protein, inefficient and error-prone repair pathways are used instead. These pathways, such as Non-homologous end joining, do not accurately repair the DNA, which can introduce mutations and genomic rearrangements. Ultimately the genome is not repaired faithfully and the predisposition to cancer greatly increases. In addition to their contribution to DNA repair, the BRCA genes have been shown to have transcriptional activity, and this functional role can also be a driving factor behind the tumor suppressor activity. Robustness is the ability of a complex system to sustain viability despite perturbations to it. In the context of a complex disease such as cancer, robustness gives cancers the ability to sustain uncontrollable growth and invasiveness despite treatments such as chemotherapy that attempt to eliminate the tumor. A complex system is robust however can be fragile to perturbations that the system not optimized against. In cancers, these fragilities have the potential to be cancer specific targets that can eradicate the disease specifically. Patients with mutations in BRCA tend to have breast and ovarian cancers that are difficult to treat; chemotherapy is the only option and no targeted therapies are available. Targeting the synthetic lethal interaction (SLI), a mechanism of robustness, between BRCA and PARP1 genes was clinically effective in treating BRCA-mutated breast and ovarian cancers. This suggests that understanding robustness in cancers can reveal potential cancer specific therapies. In this thesis, a computational approach was developed to identify candidate mechanisms of robustness in BRCA-mutated breast and ovarian cancers using the publicly accessible patient gene expression and mutation data from the Cancer Genome Atlas (TCGA). Results showed that in ovarian cancer patients with a BRCA2 mutation, the expression of genes that function in the DNA damage response were kept at stable expression state compared to those patients without a mutation. The stable expression of genes in the DNA damage response may highlight a SLI gene network that is precisely controlled. This result is significant as disrupting this precision can potentially lead to cancer specific death. In breast cancers, genes that were differentially expressed in patients with BRCA mutations were identified. A Bayesian network was performed to infer candidate interactions between BRCA1 and BRCA2 and the differentially expressed FLT3, HOXA11, HPGD, MLF1, NGFR, PLAT, and ZBTB16 genes. These genes function in processes important to cancer progression such as apoptosis and cell migration. The connection between these genes with BRCA may highlight how the BRCA genes influence cancer progression. Taken together, the findings of this thesis enhance our understanding of the BRCA genes and their role in DNA damage response and transcriptional regulation in human breast and ovarian cancers. These results have been attained from systems-level models to identify candidate mechanisms underlying robustness of cancers. The work presented predicts interesting candidate genes that may have potential as drug targets or biomarkers in BRCA-mutated breast and ovarian cancers

Changes in gene expression variability reveal a stable synthetic lethal interaction network in BRCA2-ovarian cancers

Synthetic lethal interactions (SLIs) are robust mechanisms that provide cells with the ability to remain viable despite having mutations in genes critical to the DNA damage response, a core cellular process. Studies in model organisms such as S. cerevisiae showed that thousands of genes important in maintaining DNA integrity cooperated in a SLI network. Two genes participate in a SLI when a mutation in one gene has no effect on the cell, but mutations in both interacting genes are lethal. Furthermore in C. elegans, a mutation in a critical gene that is important for development induced a change in expression variability in the synthetic lethal interactor. In cancer, targeting SLIs shows promise in selectively killing cancer cells. For example, targeting PARP1 is an effective treatment for BRCA1/2- breast and ovarian cancers. Although PARP1 is already identified as having a SLI with BRCA1/2-, computationally searching for other genes that cooperate in the SLI network could highlight genes that may have promise for being a cancer-specific drug target. Using RNA sequencing data for ovarian cancer patients with BRCA2 mutations and the R Bioconductor package pathVar, we showed that genes whose expression changes to an invariant, stable expression state are likely candidates for SLIs with BRCA2. Our results highlight the interactions between the genes with predicted SLIs and protein-coding genes that are functionally important in the DNA damage response. The method of analyzing expression variability to computationally identify genes with SLIs can be applied to query SLIs in other tumor types

Automatic LRT ticketing system

The proposed thesis is about an Automatic LRT Ticketing System. This new system uses infrared technology in a card reader. An MC68HC11 microcontroller is used to synchronize the entire operation. The LRT (Light Railway Transit) passengers will just have to purchase these cards from the cashier. The card has a predetermined value from which the fare will be debited. Each time a passenger uses the card, the fare will be debited (by punching holes on the card) from the total value of the card

CRISPR activation and interference screens decode stimulation responses in primary human T cells.

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies

Protracted neuronal recruitment in the temporal lobes of young children.

The temporal lobe of the human brain contains the entorhinal cortex (EC). This region of the brain is a highly interconnected integrative hub for sensory and spatial information; it also has a key role in episodic memory formation and is the main source of cortical hippocampal inputs1-4. The human EC continues to develop during childhood5, but neurogenesis and neuronal migration to the EC are widely considered to be complete by birth. Here we show that the human temporal lobe contains many young neurons migrating into the postnatal EC and adjacent regions, with a large tangential stream persisting until the age of around one year and radial dispersal continuing until around two to three years of age. By contrast, we found no equivalent postnatal migration in rhesus macaques (Macaca mulatta). Immunostaining and single-nucleus RNA sequencing of ganglionic&nbsp;eminence germinal zones, the EC stream and the postnatal EC revealed that most migrating cells in the EC stream are derived from the caudal ganglionic eminence and become LAMP5+RELN+ inhibitory interneurons. These late-arriving interneurons could continue to shape the processing of sensory and spatial information well into postnatal life, when children are actively interacting with their environment. The EC is one of the first regions of the brain to be affected in Alzheimers disease, and previous work has linked cognitive decline to the loss of LAMP5+RELN+ cells6,7. Our investigation reveals that many of these cells arrive in the EC through a major postnatal migratory stream in early childhood

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T&nbsp;cells.

CRISPR-Cas9 genome editing has enabled advanced T&nbsp;cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T&nbsp;cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T&nbsp;cells. T&nbsp;cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T&nbsp;cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T&nbsp;cell engineering that mitigates this genotoxicity in the clinic