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1.
Nat Methods ; 19(1): 81-89, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34949807

RESUMO

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Infecções por HIV/genética , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Proteína 9 Associada à CRISPR/genética , Movimento Celular/genética , Células Cultivadas , DNA , Técnicas de Inativação de Genes , Infecções por HIV/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , RNA Guia de Cinetoplastídeos , Proteína 1 com Domínio SAM e Domínio HD/genética , Transgenes , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
2.
Infection ; 50(3): 635-642, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34716901

RESUMO

PURPOSE: To investigate the expression of the receptor protein ACE-2 alongside the urinary tract, urinary shedding and urinary stability of SARS-CoV-2 RNA. METHODS: Immunohistochemical staining was performed on tissue from urological surgery of 10 patients. Further, patients treated for coronavirus disease (COVID-19) at specialized care-units of a university hospital were assessed for detection of SARS-CoV-2 RNA in urinary samples via PCR, disease severity (WHO score), inflammatory response of patients. Finally, the stability of SARS-CoV-2 RNA in urine was analyzed. RESULTS: High ACE-2 expression (3/3) was observed in the tubules of the kidney and prostate glands, moderate expression in urothelial cells of the bladder (0-2/3) and no expression in kidney glomeruli, muscularis of the bladder and stroma of the prostate (0/3). SARS-CoV-2 RNA was detected in 5/199 urine samples from 64 patients. Viral RNA was detected in the first urinary sample of sequential samples. Viral RNA load from other specimen as nasopharyngeal swabs (NPS) or endotracheal aspirates revealed higher levels than from urine. Detection of SARS-CoV-2 RNA in urine was not associated with impaired WHO score (median 5, range 3-8 vs median 4, range 1-8, p = 0.314), peak white blood cell count (median 24.1 × 1000/ml, range 5.19-48.1 versus median 11.9 × 1000/ml, range 2.9-60.3, p = 0.307), peak CRP (median 20.7 mg/dl, 4.2-40.2 versus median 11.9 mg/dl, range 0.1-51.9, p = 0.316) or peak IL-6 levels (median: 1442 ng/ml, range 26.7-3918 versus median 140 ng/ml, range 3.0-11,041, p = 0.099). SARS-CoV-2 RNA was stable under different storage conditions and after freeze-thaw cycles. CONCLUSIONS: SARS-CoV-2 RNA in the urine of COVID-19 patients occurs infrequently. The viral RNA load and dynamics of SARS-CoV-2 RNA shedding suggest no relevant route of transmission through the urinary tract.


Assuntos
COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Sistema Urinário , COVID-19/diagnóstico , Humanos , Masculino , RNA Viral , SARS-CoV-2/genética , Sistema Urinário/química , Eliminação de Partículas Virais
3.
Euro Surveill ; 26(43)2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34713795

RESUMO

BackgroundIn the SARS-CoV-2 pandemic, viral genomes are available at unprecedented speed, but spatio-temporal bias in genome sequence sampling precludes phylogeographical inference without additional contextual data.AimWe applied genomic epidemiology to trace SARS-CoV-2 spread on an international, national and local level, to illustrate how transmission chains can be resolved to the level of a single event and single person using integrated sequence data and spatio-temporal metadata.MethodsWe investigated 289 COVID-19 cases at a university hospital in Munich, Germany, between 29 February and 27 May 2020. Using the ARTIC protocol, we obtained near full-length viral genomes from 174 SARS-CoV-2-positive respiratory samples. Phylogenetic analyses using the Auspice software were employed in combination with anamnestic reporting of travel history, interpersonal interactions and perceived high-risk exposures among patients and healthcare workers to characterise cluster outbreaks and establish likely scenarios and timelines of transmission.ResultsWe identified multiple independent introductions in the Munich Metropolitan Region during the first weeks of the first pandemic wave, mainly by travellers returning from popular skiing areas in the Alps. In these early weeks, the rate of presumable hospital-acquired infections among patients and in particular healthcare workers was high (9.6% and 54%, respectively) and we illustrated how transmission chains can be dissected at high resolution combining virus sequences and spatio-temporal networks of human interactions.ConclusionsEarly spread of SARS-CoV-2 in Europe was catalysed by superspreading events and regional hotspots during the winter holiday season. Genomic epidemiology can be employed to trace viral spread and inform effective containment strategies.


Assuntos
COVID-19 , Infecção Hospitalar , Infecção Hospitalar/epidemiologia , Genoma Viral , Genômica , Alemanha/epidemiologia , Hospitais , Humanos , Filogenia , SARS-CoV-2
4.
Cell Rep Methods ; 4(1): 100685, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38211593

RESUMO

The molecular and immunological properties of tissue-resident resting CD4 T cells are understudied due to the lack of suitable gene editing methods. Here, we describe the ex vivo culture and gene editing methodology ediTONSIL for CD4 T cells from human tonsils. Optimized CRISPR-Cas9 RNP nucleofection results in knockout efficacies of over 90% without requiring exogenous activation. Editing can be performed on multiple cell types in bulk cultures or on isolated CD4 T cells that can be labeled and reintroduced into their tissue environment. Importantly, CD4 T cells maintain their tissue-specific properties such as viability, activation state, or immunocompetence following reassembly into lymphoid aggregates. This highly efficient and versatile gene editing workflow for tonsillar CD4 T cells enables the dissection of molecular mechanisms in ex vivo cultures of human lymphoid tissue and can be adapted to other tonsil-resident cell types.


Assuntos
Linfócitos T CD4-Positivos , Tonsila Palatina , Humanos , Edição de Genes , Tecido Linfoide
5.
Cell Rep Med ; 5(4): 101483, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38579727

RESUMO

Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.


Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Linfócitos T CD4-Positivos , Receptores de IgG/metabolismo , Autoanticorpos/metabolismo , Trogocitose
6.
Front Immunol ; 12: 632154, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093525

RESUMO

The human gastric pathogen Helicobacter pylori activates human epithelial cells by a particular combination of mechanisms, including NOD1 and ALPK1-TIFA activation. These mechanisms are characterized by a strong participation of the bacterial cag pathogenicity island, which forms a type IV secretion system (CagT4SS) that enables the bacteria to transport proteins and diverse bacterial metabolites, including DNA, glycans, and cell wall components, into human host cells. Building on previous findings, we sought to determine the contribution of lipopolysaccharide inner core heptose metabolites (ADP-heptose) in the activation of human phagocytic cells by H. pylori. Using human monocyte/macrophage-like Thp-1 cells and human primary monocytes and macrophages, we were able to determine that a substantial part of early phagocytic cell activation, including NF-κB activation and IL-8 production, by live H. pylori is triggered by bacterial heptose metabolites. This effect was very pronounced in Thp-1 cells exposed to bacterial purified lysates or pure ADP-heptose, in the absence of other bacterial MAMPs, and was significantly reduced upon TIFA knock-down. Pure ADP-heptose on its own was able to strongly activate Thp-1 cells and human primary monocytes/macrophages. Comprehensive transcriptome analysis of Thp-1 cells co-incubated with live H. pylori or pure ADP-heptose confirmed a signature of ADP-heptose-dependent transcript activation in monocyte/macrophages. Bacterial enzyme-treated lysates (ETL) and pure ADP-heptose-dependent activation differentiated monocytes into macrophages of predominantly M1 type. In Thp-1 cells, the active CagT4SS was less required for the heptose-induced proinflammatory response than in epithelial cells, while active heptose biosynthesis or pure ADP-heptose was required and sufficient for their early innate response and NF-κB activation. The present data suggest that early activation and maturation of incoming and resident phagocytic cells (monocytes, macrophages) in the H. pylori-colonized stomach strongly depend on bacterial LPS inner core heptose metabolites, also with a significant contribution of an active CagT4SS.


Assuntos
Ilhas Genômicas/fisiologia , Helicobacter pylori/metabolismo , Heptoses/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vias Biossintéticas , Helicobacter pylori/patogenicidade , Humanos , Imunidade Inata , Lipopolissacarídeos/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Monócitos/metabolismo , Transdução de Sinais , Transcriptoma , Sistemas de Secreção Tipo IV/genética , Sistemas de Secreção Tipo IV/metabolismo
7.
Cell Rep ; 37(13): 110169, 2021 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-34932974

RESUMO

The importance of pre-existing immune responses to seasonal endemic coronaviruses (HCoVs) for the susceptibility to SARS-CoV-2 infection and the course of COVID-19 is the subject of an ongoing scientific debate. Recent studies postulate that immune responses to previous HCoV infections can either have a slightly protective or no effect on SARS-CoV-2 pathogenesis and, consequently, be neglected for COVID-19 risk stratification. Challenging this notion, we provide evidence that pre-existing, anti-nucleocapsid antibodies against endemic α-coronaviruses and S2 domain-specific anti-spike antibodies against ß-coronavirus HCoV-OC43 are elevated in patients with COVID-19 compared to pre-pandemic donors. This finding is particularly pronounced in males and in critically ill patients. Longitudinal evaluation reveals that antibody cross-reactivity or polyclonal stimulation by SARS-CoV-2 infection are unlikely to be confounders. Thus, specific pre-existing immunity to seasonal coronaviruses may increase susceptibility to SARS-CoV-2 and predispose individuals to an adverse COVID-19 outcome, guiding risk management and supporting the development of universal coronavirus vaccines.


Assuntos
COVID-19/imunologia , Coronavirus/imunologia , SARS-CoV-2/imunologia , Adulto , Anticorpos/imunologia , Anticorpos Antivirais/imunologia , COVID-19/etiologia , Infecções por Coronavirus/imunologia , Coronavirus Humano OC43/imunologia , Coronavirus Humano OC43/patogenicidade , Reações Cruzadas/imunologia , Feminino , Alemanha , Humanos , Imunidade Humoral/imunologia , Imunoglobulina G/imunologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Pandemias , SARS-CoV-2/patogenicidade , Estações do Ano , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/imunologia
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