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1.
PLoS One ; 17(2): e0263563, 2022.
Article in English | MEDLINE | ID: covidwho-1793526

ABSTRACT

Deletions frequently occur in the six accessory genes of SARS-CoV-2, but most genomes with deletions are sporadic and have limited spreading capability. Here, we analyze deletions in the ORF7a of the N.7 lineage, a unique Uruguayan clade from the Brazilian B.1.1.33 lineage. Thirteen samples collected during the early SARS-CoV-2 wave in Uruguay had deletions in the ORF7a. Complete genomes were obtained by Illumina next-generation sequencing, and deletions were confirmed by Sanger sequencing and capillary electrophoresis. The N.7 lineage includes several individuals with a 12-nucleotide deletion that removes four amino acids of the ORF7a. Notably, four individuals underwent an additional 68-nucleotide novel deletion that locates 44 nucleotides downstream in the terminal region of the same ORF7a. The simultaneous occurrence of the 12 and 68-nucleotide deletions fuses the ORF7a and ORF7b, two contiguous accessory genes that encode transmembrane proteins with immune-modulation activity. The fused ORF retains the signal peptide and the complete Ig-like fold of the 7a protein and the transmembrane domain of the 7b protein, suggesting that the fused protein plays similar functions to original proteins in a single format. Our findings evidence the remarkable dynamics of SARS-CoV-2 and the possibility that single and consecutive deletions occur in accessory genes and promote changes in the genomic organization that help the virus explore genetic variations and select for new, higher fit changes.


Subject(s)
COVID-19/virology , Cell Lineage , Gene Deletion , Genome, Viral , Open Reading Frames/genetics , SARS-CoV-2/genetics , Viral Proteins/genetics , Adult , Aged , COVID-19/epidemiology , COVID-19/genetics , Child , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Middle Aged , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/isolation & purification , Uruguay/epidemiology
2.
Gastroenterology ; 161(3): 785-791, 2021 09.
Article in English | MEDLINE | ID: covidwho-1626143

ABSTRACT

Pancreatic ductal adenocarcinoma remains a major challenge in cancer medicine. Given the increase in incidence and mortality, interdisciplinary research is necessary to translate basic knowledge into therapeutic strategies improving the outcome of patients. On the 4th and 5th of February 2021, three German pancreatic cancer research centers, the Clinical Research Unit 5002 from Göttingen, the Collaborative Research Center 1321 from Munich, and Clinical Research Unit 325 from Marburg organized the 1st Virtual Göttingen-Munich-Marburg Pancreatic Cancer Meeting in order to foster scientific exchange. This report summarizes current research and proceedings presented during that meeting.


Subject(s)
Biomedical Research/trends , Pancreatic Neoplasms , Animals , Biomarkers, Tumor/genetics , COVID-19 , Cell Lineage , Diffusion of Innovation , Genetic Predisposition to Disease , Humans , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/therapy , Tumor Microenvironment , Videoconferencing
3.
Ann N Y Acad Sci ; 1506(1): 74-97, 2021 12.
Article in English | MEDLINE | ID: covidwho-1612914

ABSTRACT

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.


Subject(s)
Cell Differentiation/physiology , Cellular Reprogramming/physiology , Congresses as Topic/trends , Embryonic Development/physiology , Research Report , Single-Cell Analysis/trends , Animals , Cell Lineage/physiology , Humans , Macrophages/physiology , Single-Cell Analysis/methods
4.
Inflamm Res ; 71(1): 131-140, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1544398

ABSTRACT

OBJECTIVES: The role of B cells in COVID-19, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Here, we describe the novel landscape of circulating double-negative (DN) CD27- IgD- B cells in COVID-19 patients, representing a group of atypical and neglected subpopulations of this cell lineage. METHODS: Using multiparametric flow cytometry, we determined DN B cell subset amounts from 91 COVID-19 patients, correlated those with cytokines, clinical and laboratory parameters, and segregated them by principal components analysis. RESULTS: We detected significant increments in the DN2 and DN3 B cell subsets, while we found a relevant decrease in the DN1 B cell subpopulation, according to disease severity and patient outcomes. These DN cell numbers also appeared to correlate with pro- or anti-inflammatory signatures, respectively, and contributed to the segregation of the patients into disease severity groups. CONCLUSION: This study provides insights into DN B cell subsets' potential role in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.


Subject(s)
COVID-19/blood , COVID-19/immunology , Immunoglobulin D/blood , SARS-CoV-2 , Tumor Necrosis Factor Receptor Superfamily, Member 7/blood , Adult , Aged , Aged, 80 and over , B-Lymphocytes/cytology , COVID-19/diagnosis , COVID-19/virology , Cell Lineage , Computational Biology , Disease Progression , Female , Humans , Male , Middle Aged , Principal Component Analysis , Prognosis , Respiration, Artificial , Severity of Illness Index , Young Adult
5.
J Clin Invest ; 131(20)2021 10 15.
Article in English | MEDLINE | ID: covidwho-1470547

ABSTRACT

BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1ß have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1ß. Following IVIG treatment, activated IL-1ß+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1ß can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.


Subject(s)
COVID-19/complications , Immunoglobulins, Intravenous/therapeutic use , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/therapy , Systemic Inflammatory Response Syndrome/immunology , Systemic Inflammatory Response Syndrome/therapy , COVID-19/blood , COVID-19/immunology , COVID-19/therapy , Case-Control Studies , Cell Death/immunology , Cell Lineage/immunology , Child , Child, Preschool , Fas Ligand Protein/immunology , Female , Humans , Infant , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/blood , Leukocyte Count , Male , Mucocutaneous Lymph Node Syndrome/blood , Neutrophil Activation , Neutrophils/classification , Neutrophils/immunology , Neutrophils/pathology , Systemic Inflammatory Response Syndrome/blood
6.
Nat Commun ; 12(1): 652, 2021 01 28.
Article in English | MEDLINE | ID: covidwho-1397868

ABSTRACT

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs.


Subject(s)
Genetic Heterogeneity , Genetic Variation , Induced Pluripotent Stem Cells/metabolism , Oligodendrocyte Precursor Cells/metabolism , Single-Cell Analysis/methods , Transcriptome/genetics , Astrocytes/cytology , Astrocytes/metabolism , Cell Differentiation/genetics , Cell Line , Cell Lineage/genetics , Cholesterol/biosynthesis , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Regulatory Networks/genetics , Humans , Induced Pluripotent Stem Cells/cytology , Oligodendrocyte Precursor Cells/cytology , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Signal Transduction/genetics , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
7.
Virol Sin ; 35(3): 280-289, 2020 Jun.
Article in English | MEDLINE | ID: covidwho-1384632

ABSTRACT

Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models (ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells (CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming (CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid (2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for air-liquid interface (ALI) polarized 3D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.


Subject(s)
Cellular Reprogramming Techniques , Models, Biological , Respiratory Mucosa/cytology , Respiratory Mucosa/virology , Betacoronavirus/physiology , COVID-19 , Cell Differentiation , Cell Lineage , Cells, Cultured , Coronavirus Infections/immunology , Coronavirus Infections/virology , Epithelial Cells/cytology , Epithelial Cells/virology , Humans , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS-CoV-2
8.
Inflamm Res ; 70(8): 847-858, 2021 Aug.
Article in English | MEDLINE | ID: covidwho-1318745

ABSTRACT

BACKGROUND: Recognizing only sharp elevation in a short period of time, the COVID-19 SARS-CoV-2 propagation is more and more marked in the whole world. Induced inflammation afterwards infection engenders a high infiltration of immune cells and cytokines that triggers matrix metalloproteinases (MMPs) activation. These endopeptidases are mediators of the lung extracellular matrix (ECM), a basic element for alveoli structure and gas exchange. METHODS: When immune cells, MMPs, secreted cytokines and several other mediators are gathered a pathological matrix remodeling occurs. This phenomenon tends to tissue destruction in the first place and a pulmonary hypertrophy and fibrosis in the second place. FINDINGS: After pathological matrix remodeling establishment, pathological diseases take place even after infection state. Since post COVID-19 pulmonary fibrosis is an emerging complication of the disease, there is an urge to better understand and characterize the implication of ECM remodeling during SARS-CoV-2 infection. CONCLUSION: Targeting MMPs and their inhibitors could be a probable solution for occurred events since there are many cured patients that remain with severe sequels even after the end of infection.


Subject(s)
COVID-19/immunology , COVID-19/virology , Extracellular Matrix/metabolism , Matrix Metalloproteinases/metabolism , SARS-CoV-2 , Cell Communication , Cell Lineage , Cytokines/metabolism , Cytoplasm/metabolism , Fibrosis/immunology , Homeostasis , Humans , Hypertrophy , Immune System , Interferon-gamma/metabolism , Lung/physiopathology , Pulmonary Alveoli/metabolism , Pulmonary Fibrosis , Pulmonary Gas Exchange
9.
Emerg Microbes Infect ; 10(1): 1507-1514, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1310873

ABSTRACT

Severe respiratory disease coronavirus-2 (SARS-CoV-2) has been the most devastating disease COVID-19 in the century. One of the unsolved scientific questions of SARS-CoV-2 is the animal origin of this virus. Bats and pangolins are recognized as the most probable reservoir hosts that harbour highly similar SARS-CoV-2 related viruses (SARSr-CoV-2). This study identified a novel lineage of SARSr-CoVs, including RaTG15 and seven other viruses, from bats at the same location where we found RaTG13 in 2015. Although RaTG15 and the related viruses share 97.2% amino acid sequence identities with SARS-CoV-2 in the conserved ORF1b region, it only shows less than 77.6% nucleotide identity to all known SARSr-CoVs at the genome level, thus forming a distinct lineage in the Sarbecovirus phylogenetic tree. We found that the RaTG15 receptor-binding domain (RBD) can bind to ACE2 from Rhinolophus affinis, Malayan pangolin, and use it as an entry receptor, except for ACE2 from humans. However, it contains a short deletion and has different key residues responsible for ACE2 binding. In addition, we showed that none of the known viruses in bat SARSr-CoV-2 lineage discovered uses human ACE2 as efficiently as the pangolin-derived SARSr-CoV-2 or some viruses in the SARSr-CoV-1 lineage. Therefore, further systematic and longitudinal studies in bats are needed to prevent future spillover events caused by SARSr-CoVs or to understand the origin of SARS-CoV-2 better.


Subject(s)
Angiotensin-Converting Enzyme 2/physiology , Cell Lineage , Chiroptera/virology , SARS Virus/isolation & purification , SARS-CoV-2/classification , Animals , Host Specificity , Phylogeny , SARS Virus/classification
10.
Curr Opin Virol ; 50: 40-48, 2021 10.
Article in English | MEDLINE | ID: covidwho-1306916

ABSTRACT

The scale of the international efforts to sequence SARS-CoV-2 genomes is unprecedented. Early availability of genomes allowed rapid characterisation of the virus, thus kickstarting many highly successful vaccine development programmes. Worldwide genomic resources have provided a good understanding of the pandemic, supported close monitoring of the emergence of viral genomic diversity and pinpointed those sites to prioritise for functional characterisation. Continued genomic surveillance of global viral populations will be crucial to inform the timing of vaccine updates so as to pre-empt the spread of immune escape lineages. While genome sequencing has provided us with an exceptionally powerful tool to monitor the evolution of SARS-CoV-2, there is room for further improvements in particular in the form of less heterogeneous global surveillance and tools to rapidly identify concerning viral lineages.


Subject(s)
COVID-19/virology , Genome, Viral , SARS-CoV-2/genetics , Cell Lineage , Evolution, Molecular , Humans , Mutation
11.
Trends Genet ; 37(7): 625-630, 2021 07.
Article in English | MEDLINE | ID: covidwho-1187872

ABSTRACT

Comprehensively characterizing the cellular composition and organization of tissues has been a long-term scientific challenge that has limited our ability to study fundamental and clinical aspects of human physiology. The Human Cell Atlas (HCA) is a global collaborative effort to create a reference map of all human cells as a basis for both understanding human health and diagnosing, monitoring, and treating disease. Many aspects of the HCA are analogous to the Human Genome Project (HGP), whose completion presents a major milestone in modern biology. To commemorate the HGP's 20-year anniversary of completion, we discuss the launch of the HCA in light of the HGP, and highlight recent progress by the HCA consortium.


Subject(s)
Cell Lineage/genetics , Cell Physiological Phenomena/genetics , Cells/classification , Genome, Human/genetics , Human Genome Project , Humans
12.
Proc Natl Acad Sci U S A ; 118(22)2021 06 01.
Article in English | MEDLINE | ID: covidwho-1232098

ABSTRACT

Comprehensive and accurate comparisons of transcriptomic distributions of cells from samples taken from two different biological states, such as healthy versus diseased individuals, are an emerging challenge in single-cell RNA sequencing (scRNA-seq) analysis. Current methods for detecting differentially abundant (DA) subpopulations between samples rely heavily on initial clustering of all cells in both samples. Often, this clustering step is inadequate since the DA subpopulations may not align with a clear cluster structure, and important differences between the two biological states can be missed. Here, we introduce DA-seq, a targeted approach for identifying DA subpopulations not restricted to clusters. DA-seq is a multiscale method that quantifies a local DA measure for each cell, which is computed from its k nearest neighboring cells across a range of k values. Based on this measure, DA-seq delineates contiguous significant DA subpopulations in the transcriptomic space. We apply DA-seq to several scRNA-seq datasets and highlight its improved ability to detect differences between distinct phenotypes in severe versus mildly ill COVID-19 patients, melanomas subjected to immune checkpoint therapy comparing responders to nonresponders, embryonic development at two time points, and young versus aging brain tissue. DA-seq enabled us to detect differences between these phenotypes. Importantly, we find that DA-seq not only recovers the DA cell types as discovered in the original studies but also reveals additional DA subpopulations that were not described before. Analysis of these subpopulations yields biological insights that would otherwise be undetected using conventional computational approaches.


Subject(s)
Aging/genetics , COVID-19/genetics , Cell Lineage/genetics , Melanoma/genetics , RNA, Small Cytoplasmic/genetics , Skin Neoplasms/genetics , Aging/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/virology , Brain/cytology , Brain/metabolism , COVID-19/immunology , COVID-19/pathology , COVID-19/virology , Cell Lineage/immunology , Cytokines/genetics , Cytokines/immunology , Datasets as Topic , Dendritic Cells/immunology , Dendritic Cells/virology , Gene Expression Profiling , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , Melanoma/immunology , Melanoma/pathology , Monocytes/immunology , Monocytes/virology , Phenotype , RNA, Small Cytoplasmic/immunology , SARS-CoV-2/pathogenicity , Severity of Illness Index , Single-Cell Analysis/methods , Skin Neoplasms/immunology , Skin Neoplasms/pathology , T-Lymphocytes/immunology , T-Lymphocytes/virology , Transcriptome
13.
Emerg Microbes Infect ; 10(1): 885-893, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1201494

ABSTRACT

Mutations in the SARS-CoV-2 Membrane (M) gene are relatively uncommon. The M gene encodes the most abundant viral structural protein, and is implicated in multiple viral functions, including initial attachment to the host cell via heparin sulphate proteoglycan, viral protein assembly in conjunction with the N and E genes, and enhanced glucose transport. We have identified a recent spike in the frequency of reported SARS-CoV-2 genomes carrying M gene mutations. This is associated with emergence of a new sub-B.1 clade, B.1.I82T, defined by the previously unreported M:I82T mutation within TM3, the third of three membrane spanning helices implicated in glucose transport. The frequency of this mutation increased in the USA from 0.014% in October 2020 to 1.62% in February 2021, a 116-fold change. While constituting 0.7% of the isolates overall, M:I82T sub-B.1 lineage accounted for 14.4% of B.1 lineage isolates in February 2021, similar to the rapid initial increase previously seen with the B.1.1.7 and B.1.429 lineages, which quickly became the dominant lineages in Europe and California over a period of several months. A similar increase in incidence was also noted in another related mutation, V70L, also within the TM2 transmembrane helix. These M mutations are associated with younger patient age (4.6 to 6.3 years). The rapid emergence of this B.1.I82T clade, recently named Pangolin B.1.575 lineage, suggests that this M gene mutation is more biologically fit, perhaps related to glucose uptake during viral replication, and should be included in ongoing genomic surveillance efforts and warrants further evaluation for potentially increased pathogenic and therapeutic implications.


Subject(s)
COVID-19/virology , Mutation , SARS-CoV-2/genetics , Viral Matrix Proteins/genetics , Adult , Cell Lineage , Child , Child, Preschool , Humans , Phylogeny
14.
J Mol Cell Biol ; 13(3): 197-209, 2021 07 06.
Article in English | MEDLINE | ID: covidwho-1145182

ABSTRACT

Although millions of patients have clinically recovered from COVID-19, little is known about the immune status of lymphocytes in these individuals. In this study, the peripheral blood mononuclear cells of a clinically recovered (CR) cohort were comparatively analyzed with those of an age- and sex-matched healthy donor cohort. We found that CD8+ T cells in the CR cohort had higher numbers of effector T cells and effector memory T cells but lower Tc1 (IFN-γ+), Tc2 (IL-4+), and Tc17 (IL-17A+) cell frequencies. The CD4+ T cells of the CR cohort were decreased in frequency, especially the central memory T cell subset. Moreover, CD4+ T cells in the CR cohort showed lower programmed cell death protein 1 (PD-1) expression and had lower frequencies of Th1 (IFN-γ+), Th2 (IL-4+), Th17 (IL-17A+), and circulating follicular helper T (CXCR5+PD-1+) cells. Accordingly, the proportion of isotype-switched memory B cells (IgM-CD20hi) among B cells in the CR cohort showed a significantly lower proportion, although the level of the activation marker CD71 was elevated. For CD3-HLA-DR- lymphocytes in the CR cohort, in addition to lower levels of IFN-γ, granzyme B and T-bet, the correlation between T-bet and IFN-γ was not observed. Additionally, by taking into account the number of days after discharge, all the phenotypes associated with reduced function did not show a tendency toward recovery within 4‒11 weeks. The remarkable phenotypic alterations in lymphocytes in the CR cohort suggest that  severe acute respiratory syndrome coronavirus 2 infection profoundly affects lymphocytes and potentially results in dysfunction even after clinical recovery.


Subject(s)
CD8-Positive T-Lymphocytes/virology , COVID-19/blood , Leukocytes, Mononuclear/virology , SARS-CoV-2/pathogenicity , Adult , Aged , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , COVID-19/epidemiology , COVID-19/pathology , COVID-19/virology , Cell Lineage/genetics , Cell Lineage/immunology , Female , Gene Expression Regulation/immunology , Granzymes/genetics , Humans , Interferon-gamma/genetics , Leukocytes, Mononuclear/pathology , Male , Middle Aged , T-Box Domain Proteins/genetics , Th1 Cells/immunology , Th1 Cells/virology , Th17 Cells/immunology , Th17 Cells/virology , Th2 Cells/immunology , Th2 Cells/virology
15.
Arterioscler Thromb Vasc Biol ; 41(1): 415-429, 2021 01.
Article in English | MEDLINE | ID: covidwho-1027162

ABSTRACT

OBJECTIVE: The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis. Approach and Results: VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease and from normal aortic valves unsuitable for grafting (control VICs). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony-forming cells. A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90+/CD73+/CD44+ expression and multipotent-like differentiation ability. When VICp were cocultured with endothelial colony-forming cells, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and control VIC conditioned media were compared using serial ELISA regarding quantification of endothelial and angiogenic factors. Higher expression of VEGF (vascular endothelial growth factor)-A was observed at the protein level in VICp-conditioned media and confirmed at the mRNA level in VICp compared with control VIC. Conditioned media from VICp induced in vitro a significant increase in endothelial colony-forming cell proliferation, migration, and sprouting compared with conditioned media from control VIC. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in angiogenesis by a VEGF-A dependent mechanism. CONCLUSIONS: We provide here the first proof of an angiogenic potential of human VICs isolated from patients with calcific aortic valve disease. These results point to a novel function of VICp in valve vascularization during calcific aortic valve disease, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and VEGF-A to slow calcific aortic valve disease progression warrants further investigation.


Subject(s)
Aortic Valve Stenosis/metabolism , Aortic Valve/metabolism , Aortic Valve/pathology , Calcinosis/metabolism , Cell Differentiation , Cell Lineage , Endothelial Progenitor Cells/metabolism , Neovascularization, Pathologic , Vascular Endothelial Growth Factor A/metabolism , Adult , Aged , Aged, 80 and over , Animals , Aortic Valve Stenosis/pathology , Calcinosis/pathology , Case-Control Studies , Cells, Cultured , Coculture Techniques , Endothelial Progenitor Cells/pathology , Endothelial Progenitor Cells/transplantation , Female , Humans , Male , Mice, Nude , Middle Aged , Osteogenesis , Paracrine Communication , Phenotype , Signal Transduction , Vascular Endothelial Growth Factor A/genetics
16.
Int J Mol Sci ; 21(22)2020 Nov 11.
Article in English | MEDLINE | ID: covidwho-918209

ABSTRACT

Immune memory is a defining characteristic of adaptive immunity, but recent work has shown that the activation of innate immunity can also improve responsiveness in subsequent exposures. This has been coined "trained immunity" and diverges with the perception that the innate immune system is primitive, non-specific, and reacts to novel and recurrent antigen exposures similarly. The "exposome" is the cumulative exposures (diet, exercise, environmental exposure, vaccination, genetics, etc.) an individual has experienced and provides a mechanism for the establishment of immune training or immunotolerance. It is becoming increasingly clear that trained immunity constitutes a delicate balance between the dose, duration, and order of exposures. Upon innate stimuli, trained immunity or tolerance is shaped by epigenetic and metabolic changes that alter hematopoietic stem cell lineage commitment and responses to infection. Due to the immunomodulatory role of the exposome, understanding innate immune training is critical for understanding why some individuals exhibit protective phenotypes while closely related individuals may experience immunotolerant effects (e.g., the order of exposure can result in completely divergent immune responses). Research on the exposome and trained immunity may be leveraged to identify key factors for improving vaccination development, altering inflammatory disease development, and introducing potential new prophylactic treatments, especially for diseases such as COVID-19, which is currently a major health issue for the world. Furthermore, continued exposome research may prevent many deleterious effects caused by immunotolerance that frequently result in host morbidity or mortality.


Subject(s)
Betacoronavirus/immunology , Cell Lineage/immunology , Coronavirus Infections/immunology , Immunity, Innate/immunology , Immunologic Memory/immunology , Pneumonia, Viral/immunology , COVID-19 , DNA Methylation/genetics , Dendritic Cells/immunology , Exposome , Histone Code/genetics , Humans , Immune Tolerance/immunology , Killer Cells, Natural/immunology , Macrophages/immunology , Pandemics , SARS-CoV-2
17.
Protein Cell ; 11(10): 740-770, 2020 10.
Article in English | MEDLINE | ID: covidwho-709445

ABSTRACT

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.


Subject(s)
Aging/immunology , Betacoronavirus , Coronavirus Infections/immunology , Immune System/immunology , Pandemics , Pneumonia, Viral/immunology , Single-Cell Analysis , Adult , Aged , Aged, 80 and over , Aging/genetics , CD4-Positive T-Lymphocytes/metabolism , COVID-19 , Cell Lineage , Chromatin Assembly and Disassembly , Cytokine Release Syndrome/etiology , Cytokine Release Syndrome/immunology , Cytokines/biosynthesis , Cytokines/genetics , Disease Susceptibility , Flow Cytometry/methods , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Rearrangement , Humans , Immune System/cytology , Immune System/growth & development , Immunocompetence/genetics , Inflammation/genetics , Inflammation/immunology , Mass Spectrometry/methods , Middle Aged , SARS-CoV-2 , Sequence Analysis, RNA , Transcriptome , Young Adult
19.
Cytotherapy ; 22(8): 458-472, 2020 08.
Article in English | MEDLINE | ID: covidwho-209852

ABSTRACT

BACKGROUND AIMS: Human platelet lysate can replace fetal bovine serum (FBS) for xeno-free ex vivo expansion of mesenchymal stromal cells (MSCs), but pooling of platelet concentrates (PCs) increases risks of pathogen transmission. We evaluated the feasibility of performing nanofiltration of platelet lysates and determined the impact on expansion of bone marrow-derived MSCs. METHODS: Platelet lysates were prepared by freeze-thawing of pathogen-reduced (Intercept) PCs suspended in 65% storage solution (SPP+) and 35% plasma, and by serum-conversion of PCs suspended in 100% plasma. Lysates were added to the MSC growth media at 10% (v/v), filtered and subjected to cascade nanofiltration on 35- and 19-nm Planova filters. Media supplemented with 10% starting platelet lysates or FBS were used as the controls. Impacts of nanofiltration on the growth media composition, removal of platelet extracellular vesicles (PEVs) and MSC expansion were evaluated. RESULTS: Nanofiltration did not detrimentally affect contents of total protein and growth factors or the biochemical composition. The clearance factor of PEVs was >3 log values. Expansion, proliferation, membrane markers, differentiation potential and immunosuppressive properties of cells in nanofiltered media were consistently better than those expanded in FBS-supplemented media. Compared with FBS, chondrogenesis and osteogenesis genes were expressed more in nanofiltered media, and there were fewer senescent cells over six passages. CONCLUSIONS: Nanofiltration of growth media supplemented with two types of platelet lysates, including one prepared from pathogen-reduced PCs, is technically feasible. These data support the possibility of developing pathogen-reduced xeno-free growth media for clinical-grade propagation of human cells.


Subject(s)
Blood Platelets/cytology , Cell Culture Techniques/methods , Filtration , Mesenchymal Stem Cells/cytology , Nanotechnology , Adipogenesis/drug effects , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Lineage/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Culture Media/pharmacology , Extracellular Vesicles/metabolism , Gene Expression Profiling , Humans , Immunophenotyping , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Mesenchymal Stem Cells/drug effects , Osteogenesis/drug effects , Particle Size , Serum/chemistry
20.
Science ; 369(6499): 50-54, 2020 07 03.
Article in English | MEDLINE | ID: covidwho-154670

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.


Subject(s)
Betacoronavirus/physiology , Enterocytes/virology , Ileum/virology , Virus Replication , Angiotensin-Converting Enzyme 2 , Betacoronavirus/ultrastructure , Cell Culture Techniques , Cell Differentiation , Cell Lineage , Cell Proliferation , Culture Media , Enterocytes/metabolism , Enterocytes/ultrastructure , Gene Expression , Humans , Ileum/metabolism , Ileum/ultrastructure , Lung/virology , Male , Organoids , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Respiratory Mucosa/virology , SARS Virus/physiology , SARS-CoV-2
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