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2.
Med (N Y) ; 3(7): 468-480.e5, 2022 07 08.
Article in English | MEDLINE | ID: covidwho-1851770

ABSTRACT

BACKGROUND: Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. METHODS: We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine. FINDINGS: The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts. CONCLUSIONS: Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine. FUNDING: This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center.


Subject(s)
BNT162 Vaccine , COVID-19 , Cell-Free Nucleic Acids , SARS-CoV-2 , Adult , Aged , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , BNT162 Vaccine/administration & dosage , COVID-19/immunology , COVID-19/prevention & control , Cell-Free Nucleic Acids/genetics , Cell-Free Nucleic Acids/immunology , Female , Humans , Immunization, Secondary , Male , Memory B Cells/immunology , Memory B Cells/metabolism , Middle Aged , SARS-CoV-2/immunology , Young Adult
3.
Bull Exp Biol Med ; 172(4): 495-498, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1756827

ABSTRACT

The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.


Subject(s)
COVID-19/metabolism , Cell-Free Nucleic Acids/genetics , DNA, Mitochondrial/genetics , NADH Dehydrogenase/genetics , Real-Time Polymerase Chain Reaction/standards , Adult , Aged , COVID-19/virology , Case-Control Studies , Cell-Free Nucleic Acids/blood , DNA Primers/chemical synthesis , DNA, Mitochondrial/blood , Female , Humans , Male , Middle Aged , Mitochondria/genetics , Mitochondria/virology , NADH Dehydrogenase/blood , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/pathogenicity
4.
Oxid Med Cell Longev ; 2022: 1630918, 2022.
Article in English | MEDLINE | ID: covidwho-1714452

ABSTRACT

BACKGROUND: The impairment of microcirculation is associated with the unfavorable outcome for extracorporeal membrane oxygenation (ECMO) patients. Studies revealed that pulsatile modification improves hemodynamics and attenuates inflammation during ECMO support. However, whether flow pattern impacts microcirculation and endothelial integrity is rarely documented. The objective of this work was to explore how pulsatility affects microcirculation during ECMO. METHODS: Canine animal models with cardiac arrest were supported by ECMO, with the i-Cor system used to generate nonpulsatile or pulsatile flow. The sublingual microcirculation parameters were examined using the CytoCam microscope system. The expression of hsa_circ_0007367, a circular RNA, was measured during ECMO support. In vitro validation was performed in pulmonary vascular endothelial cells (PMVECs) exposed to pulsatile or nonpulsatile flow, and the expressions of hsa_circ_0007367, endothelial tight junction markers, endothelial adhesive molecules, endothelial nitric oxide synthases (eNOS), and NF-κB signaling activity were analyzed. RESULTS: The pulsatile modification of ECMO enhanced microcirculatory perfusion, attenuated pulmonary inflammation, and stabilized endothelial integrity in animal models; meanwhile, the expression of hsa_circ_0007367 was significantly upregulated both in animals and PMVECs exposed to pulsatile flow. In particular, upregulation of hsa_circ_0007367 stabilized the expressions of endothelial tight junction markers zonula occludens- (ZO-) 1 and occludin, followed by modulating the endothelial nitric oxide synthases (eNOS) activity and inhibiting the NF-κB signaling pathway. CONCLUSION: The modification of pulsatility contributes to microcirculatory perfusion and endothelial integrity during ECMO. The expression of hsa_circ_0007367 plays a pivotal role in this protective mechanism.


Subject(s)
Cell-Free Nucleic Acids/genetics , Endothelial Cells/physiology , Extracorporeal Membrane Oxygenation/methods , Heart Arrest/therapy , Animals , Cell Adhesion Molecules/metabolism , Cells, Cultured , Dogs , Endothelial Cells/metabolism , Heart Arrest/genetics , Heart Arrest/pathology , Heart Arrest/physiopathology , Inflammation , Lung/blood supply , Lung/pathology , Microcirculation , Nitric Oxide Synthase Type III/metabolism , Occludin/genetics , Occludin/metabolism , Pulsatile Flow , Rats , Zonula Occludens-1 Protein/genetics , Zonula Occludens-1 Protein/metabolism
5.
J Clin Invest ; 131(7)2021 04 01.
Article in English | MEDLINE | ID: covidwho-1076050

ABSTRACT

BACKGROUNDCirculating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA may represent a more reliable indicator of infection than nasal RNA, but quantitative reverse transcription PCR (RT-qPCR) lacks diagnostic sensitivity for blood samples.METHODSA CRISPR-augmented RT-PCR assay that sensitively detects SARS-CoV-2 RNA was employed to analyze viral RNA kinetics in longitudinal plasma samples from nonhuman primates (NHPs) after virus exposure; to evaluate the utility of blood SARS-CoV-2 RNA detection for coronavirus disease 2019 (COVID-19) diagnosis in adults cases confirmed by nasal/nasopharyngeal swab RT-PCR results; and to identify suspected COVID-19 cases in pediatric and at-risk adult populations with negative nasal swab RT-qPCR results. All blood samples were analyzed by RT-qPCR to allow direct comparisons.RESULTSCRISPR-augmented RT-PCR consistently detected SARS-CoV-2 RNA in the plasma of experimentally infected NHPs from 1 to 28 days after infection, and these increases preceded and correlated with rectal swab viral RNA increases. In a patient cohort (n = 159), this blood-based assay demonstrated 91.2% diagnostic sensitivity and 99.2% diagnostic specificity versus a comparator RT-qPCR nasal/nasopharyngeal test, whereas RT-qPCR exhibited 44.1% diagnostic sensitivity and 100% specificity for the same blood samples. This CRISPR-augmented RT-PCR assay also accurately identified patients with COVID-19 using one or more negative nasal swab RT-qPCR results.CONCLUSIONResults of this study indicate that sensitive detection of SARS-CoV-2 RNA in blood by CRISPR-augmented RT-PCR permits accurate COVID-19 diagnosis, and can detect COVID-19 cases with transient or negative nasal swab RT-qPCR results, suggesting that this approach could improve COVID-19 diagnosis and the evaluation of SARS-CoV-2 infection clearance, and predict the severity of infection.TRIAL REGISTRATIONClinicalTrials.gov. NCT04358211.FUNDINGDepartment of Defense, National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, and the National Center for Research Resources.


Subject(s)
COVID-19/blood , COVID-19/virology , Cell-Free Nucleic Acids/blood , Cell-Free Nucleic Acids/genetics , RNA, Viral/blood , RNA, Viral/genetics , SARS-CoV-2 , Adolescent , Adult , Aged , Animals , COVID-19/diagnosis , COVID-19 Nucleic Acid Testing/methods , COVID-19 Nucleic Acid Testing/statistics & numerical data , CRISPR-Cas Systems , Child , Child, Preschool , Disease Models, Animal , Female , Humans , Infant , Longitudinal Studies , Macaca mulatta , Male , Middle Aged , Pandemics , SARS-CoV-2/genetics , Sensitivity and Specificity , Time Factors
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