The effective treatment of acute myeloid leukemia (AML) is very challenging. Due to the immense heterogeneity of this disease, treating it using a "one size fits all" approach is ineffective and only ...benefits a subset of patients. Instead, there is a shift towards more personalized treatment based on the patients' genomic signature. This shift has facilitated the increased revelation of novel insights into pathways that lead to the survival and propagation of AML cells. These AML survival pathways are involved in drug resistance, evasion of the immune system, reprogramming metabolism, and impairing differentiation. In addition, based on the reports of enhanced clinical efficiencies when combining drugs or treatments, deeper investigation into possible pathways, which can be targeted together to increase treatment response in a wider group of patients, is warranted. In this review, not only is a comprehensive summary of targets involved in these pathways provided, but also insights into the potential of targeting these molecules in combination therapy will be discussed.
A versatile portfolio of diagnostic tests is essential for the containment of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. Besides nucleic acid-based test systems ...and point-of-care (POCT) antigen (Ag) tests, quantitative, laboratory-based nucleocapsid Ag tests for SARS-CoV-2 have recently been launched. Here, we evaluated four commercial Ag tests on automated platforms and one POCT to detect SARS-CoV-2. We evaluated PCR-positive (
n
= 107) and PCR-negative (
n
= 303) respiratory swabs from asymptomatic and symptomatic patients at the end of the second pandemic wave in Germany (February–March 2021) as well as clinical isolates EU1 (B.1.117), variant of concern (VOC) Alpha (B.1.1.7) or Beta (B.1.351), which had been expanded in a biosafety level 3 laboratory. The specificities of automated SARS-CoV-2 Ag tests ranged between 97.0 and 99.7% (Lumipulse G SARS-CoV-2 Ag (Fujirebio): 97.03%, Elecsys SARS-CoV-2 Ag (Roche Diagnostics): 97.69%; LIAISON
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SARS-CoV-2 Ag (Diasorin) and SARS-CoV-2 Ag ELISA (Euroimmun): 99.67%). In this study cohort of hospitalized patients, the clinical sensitivities of tests were low, ranging from 17.76 to 52.34%, and analytical sensitivities ranged from 420,000 to 25,000,000 Geq/ml. In comparison, the detection limit of the Roche Rapid Ag Test (RAT) was 9,300,000 Geq/ml, detecting 23.58% of respiratory samples. Receiver-operating-characteristics (ROCs) and Youden’s index analyses were performed to further characterize the assays’ overall performance and determine optimal assay cutoffs for sensitivity and specificity. VOCs carrying up to four amino acid mutations in nucleocapsid were detected by all five assays with characteristics comparable to non-VOCs. In summary, automated, quantitative SARS-CoV-2 Ag tests show variable performance and are not necessarily superior to a standard POCT. The efficacy of any alternative testing strategies to complement nucleic acid-based assays must be carefully evaluated by independent laboratories prior to widespread implementation.
Since autumn 2020, rapid antigen tests (RATs) have been implemented in several countries as an important pillar of the national testing strategy to rapidly screen for infections on site during the ...SARS-CoV-2 pandemic. The current surge in infection rates around the globe is driven by the variant of concern (VoC) omicron (B.1.1.529). Here, we evaluated the performance of nine SARS-CoV-2 RATs in a single-centre laboratory study. We examined a total of 115 SARS-CoV-2 PCR-negative and 166 SARS-CoV-2 PCR-positive respiratory swab samples (101 omicron, 65 delta (B.1.617.2)) collected from October 2021 until January 2022 as well as cell culture-expanded clinical isolates of both VoCs. In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 1.77 × 10
6
to 7.03 × 10
7
RNA copies subjected to the RAT for omicron compared to 1.32 × 10
5
to 2.05 × 10
6
for delta. To score positive in these point-of-care tests, up to 10-fold (LoD50) or 101-fold (LoD95) higher virus loads were required for omicron- compared to delta-containing samples. The rates of true positive test results for omicron samples in the highest virus load category (Ct values < 25) ranged between 31.4 and 77.8%, while they dropped to 0–8.3% for samples with intermediate Ct values (25–30). Of note, testing of expanded virus stocks suggested a comparable RAT sensitivity of both VoCs, questioning the predictive value of this type of in vitro-studies for clinical performance. Given their importance for national test strategies in the current omicron wave, awareness must be increased for the reduced detection rate of omicron infections by RATs and a short list of suitable RATs that fulfill the minimal requirements of performance should be rapidly disclosed.
Successful containment strategies for the SARS-CoV-2 pandemic will depend on reliable diagnostic assays. Point-of-care antigen tests (POCT) may provide an alternative to time-consuming PCR tests to ...rapidly screen for acute infections on site. Here, we evaluated two SARS-CoV-2 antigen tests: the STANDARD™ F COVID-19 Ag FIA (FIA) and the SARS-CoV-2 Rapid Antigen Test (RAT). For diagnostic assessment, we used a large set of PCR-positive and PCR-negative respiratory swabs from asymptomatic and symptomatic patients and health care workers in the setting of two University Hospitals in Munich, Germany, i.e. emergency rooms, patient care units or employee test centers. For FIA, overall clinical sensitivity and specificity were 45.4% (
n
= 381) and 97.8% (
n
= 360), respectively, and for RAT, 50.3% (
n
= 445) and 97.7% (
n
= 386), respectively. For primary diagnosis of asymptomatic and symptomatic individuals, diagnostic sensitivities were 60.9% (FIA) (
n
= 189) and 64.5% (RAT) (
n
= 256). This questions these tests’ utility for the reliable detection of acute SARS-CoV-2-infected individuals, in particular in high-risk settings. We support the proposal that convincing high-quality outcome data on the impact of false-negative and false-positive antigen test results need to be obtained in a POCT setting. Moreover, the efficacy of alternative testing strategies to complement PCR assays must be evaluated by independent laboratories, prior to widespread implementation in national and international test strategies.
Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a ...purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine–aspartate domain–containing protein 1 (SAMHD1) ex vivo in resting CD4+ T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4+ T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx.
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•Optimized lentiviral Vpx eukaryotic expression construct that preserved functionality for SAMHD1 degradation.•Established good conditions to optimize lentiviral Vpx purification and decrease impurities from HEK293T cells.•Established an ex vivo assay to monitor Vpx’ ability to degrade SAMHD1 in intact cells.
The nucleoside analog cytarabine (Ara-C) is an essential component of primary and salvage chemotherapy regimens for acute myeloid leukemia (AML). After cellular uptake, Ara-C is converted into its ...therapeutically active triphosphate metabolite, Ara-CTP, which exerts antileukemic effects, primarily by inhibiting DNA synthesis in proliferating cells. Currently, a substantial fraction of patients with AML fail to respond effectively to Ara-C therapy, and reliable biomarkers for predicting the therapeutic response to Ara-C are lacking. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that cleaves physiological dNTPs into deoxyribonucleosides and inorganic triphosphate. Although it has been postulated that SAMHD1 sensitizes cancer cells to nucleoside-analog derivatives through the depletion of competing dNTPs, we show here that SAMHD1 reduces Ara-C cytotoxicity in AML cells. Mechanistically, dGTP-activated SAMHD1 hydrolyzes Ara-CTP, which results in a drastic reduction of Ara-CTP in leukemic cells. Loss of SAMHD1 activity-through genetic depletion, mutational inactivation of its triphosphohydrolase activity or proteasomal degradation using specialized, virus-like particles-potentiates the cytotoxicity of Ara-C in AML cells. In mouse models of retroviral AML transplantation, as well as in retrospective analyses of adult patients with AML, the response to Ara-C-containing therapy was inversely correlated with SAMHD1 expression. These results identify SAMHD1 as a potential biomarker for the stratification of patients with AML who might best respond to Ara-C-based therapy and as a target for treating Ara-C-refractory AML.
CD4＋ T lymphocytes represent the main target cell population of human immunodeficiency virus （HIV）. In an activated state, CD4＋ T cells residing in lymphoid organs are a major reservoir of ongoing ...HIV-1 replication in in- fected individuals. In contrast, resting CD4＋ T cells are highly resistant to productive HIV-1 infection, yet are mas- sively depleted during disease progression and represent a substantial latent reservoir for the virus in vivo. Barriers preventing replication of HIV-1 in resting CD4＋ T cells include a rigid layer of cortical actin and, early after HIV-1 entry, a block that limits reverse transcription of incoming viral RNA genomes. Defining the molecular bases of these restrictions has remained one of the central open questions in HIV research. Recent advances unraveled mechanisms by which HIV-1 bypasses the entry block and established the host cell restriction factor SAMHDI, a deoxynucleoside tripbosphate triphosphohydrolase, as a central determinant of the cellular restriction to HIV-1 reverse transcription in resting CD4＋ T cells. This review summarizes our current molecular and pathophysiological understanding of the mnlti-faceted interactions of HIV-1 with resting CD4＋ T lymphocytes.
Interferon-inducible transmembrane proteins (IFITMs) are a family of transmembrane proteins. The subgroup of immunity-related (IR-)IFITMs is involved in adaptive and innate immune responses, being ...especially active against viruses. Here, we suggest that IFITMs should be classified as (1) a canonical IFITM gene cluster, which is located on the same chromosome, and (2) IFITM retrogenes, with a random and unique location at different positions within the genome. Phylogenetic analyses of the canonical cluster revealed the existence of three novel groups of primate IFITMs (pIFITM) in the IR-IFITM clade: the prosimian pIFITMs(pro), the new world monkey pIFITMs(nwm) and the old world monkey pIFITMs(owm). Therefore, we propose a new nomenclature: IR-pIFITM1, IR-pIFITM2, IR-pIFITM3, IR-pIFITMnwm, IR-pIFITMowm, and IR-pIFITMpro. We observed divergent evolution for pIFITM5 and pIFITM10, and evidence for concerted evolution and a mechanism of birth-and-death evolution model for the IR-pIFITMs. In contrast, the IFITMs scattered throughout the genomes possessed features of retrogenes retrotransposed by class 1 transposable elements. The origin of the IFITM retrogenes correspond to more recent events. We hypothesize that the transcript of a canonical IFITM3 has been constantly retrotransposed using class 1 transposable elements resulting in the IFITM retro(pseudo)genes. The unique pattern of each species has most likely been caused by constant pseudogenization and loss of the retro(pseudo)genes. This suggests a third mechanism of evolution for the IR-IFITMs in primates, similar to the birth-and-death model of evolution, but via a transposable element mechanism, which resulted in retro(pseudo)genes.
Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral ...genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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