Identification of cytokine-regulated genes in human leukocytes in vivo

Acquired immunodeficiency syndrome (AIDS) is a worldwide disease characterized by impairments of immune function. AIDS can be associated with oxidative stress (OS) that can be linked to selenium (Se) deficiency. Se is fundamental for the synthesis of selenoproteins, such as glutathione peroxidase and thioredoxin reductase. These enzymes catalyze the decomposition of reactive oxygen species and contribute to maintain equilibrium in cell redox status. Literature data indicate that organoselenium compounds, such as ebselen and diphenyl diselenide, have antioxidant properties in vitro and in vivo models associated with OS. Nevertheless, selenocompounds can also react and oxidize thiols groups, inducing toxicity in mammals. Here, we tested the potential cytotoxic and genotoxic properties of six analogs of the prototypal anti-HIV drug azidothymidine (AZT) containing Se (5′-Se-(phenyl)zidovudine; 5′-Se-(1,3,5-trimethylphenyl)zidovudine; 5′-Se-(1-naphtyl)zidovudine; 5′-Se-(4-chlorophenyl)zidovudine) (C4); 5′-Se-(4-methylphenyl)zidovudine (C5); and 5′-(4-methylbenzoselenoate)zidovudine). C5 increased the rate of dithiothreitol oxidation (thiol oxidase activity) and C2-C4 and C6 (at 100 µM) increased DNA damage index (DI) in human leukocytes. Moreover, C5 (200 µM) decreased human leukocyte viability to about 50%. Taken together, these results indicated the low in vitro toxicity in human leukocytes of some Se-containing analogs of AZT.

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Studies on Agranulocytosis. II. The Effect of Chlorpromazine upon the Influx of S35 L-Cystine and L-Methionine into Human Leukocytes

Blood ◽

10.1182/blood.v16.4.1456.1456 ◽

1960 ◽

Vol 16 (4) ◽

pp. 1456-1468 ◽

Cited By ~ 1

Author(s):

ANTHONY V. PISCIOTTA ◽

SHIRLEY N. EBBE ◽

MARY DALY ◽

MONA RUWALDT ◽

MILTON GLASER ◽

...

Keyword(s):

Whole Blood ◽

Blood Cells ◽

The Other ◽

In Vivo Studies ◽

Human Leukocytes ◽

In Vivo Administration

Abstract 1. When whole blood was incubated in vitro with S-35 L-cystine and L-methionine, the blood cells became radioactive. 2. Preincubation of whole blood from normals and from patients susceptible to agranulocytosis with chlorpromazine showed no effect upon uptake of S-35 L-cystine and L-methionine by leukocytes. 3. The in vivo administration of S-35 L-cystine was followed by the appearance of radioactive leukocytes. Peak radioactivity occurred in leukocytes in 5 to 12 days. 4. Pretreatment of test subjects with large doses of chlorpromazine did not block the uptake of S-35 L-cystine by leukocytes in vivo. Leukocytes of women showed an increase in the incorporation of S-35 L-cystine, in vivo. Studies performed in vivo on two persons during recovery from agranulocytosis showed enhanced uptake of L-cystine in one and a normal uptake in the other.

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Two-photon excited endogenous fluorescence for label-free in vivo imaging ingestion of disease-causing bacteria by human leukocytes

10.1117/12.2002768 ◽

2013 ◽

Author(s):

Yan Zeng ◽

Bo Yan ◽

Qiqi Sun ◽

Seng Khoon Teh ◽

Wei Zhang ◽

...

Keyword(s):

In Vivo Imaging ◽

Label Free ◽

Human Leukocytes ◽

Two Photon ◽

Endogenous Fluorescence

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Ethoxy acetalated dextran-based nanocarriers accomplish efficient inhibition of leukotriene formation by a novel FLAP antagonist in human leukocytes and blood

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-04039-7 ◽

2021 ◽

Author(s):

Christian Kretzer ◽

Blerina Shkodra ◽

Paul Klemm ◽

Paul M. Jordan ◽

Daniel Schröder ◽

...

Keyword(s):

Light Scattering ◽

Whole Blood ◽

Degradation Kinetics ◽

Drug Loading ◽

Biological Assays ◽

Human Leukocytes ◽

Vis Spectroscopy ◽

Chemical Attributes ◽

Size And Morphology

AbstractLeukotrienes are pro-inflammatory lipid mediators generated by 5-lipoxygenase aided by the 5-lipoxygenase-activating protein (FLAP). BRP-201, a novel benzimidazole-based FLAP antagonist, inhibits leukotriene biosynthesis in isolated leukocytes. However, like other FLAP antagonists, BRP-201 fails to effectively suppress leukotriene formation in blood, which limits its therapeutic value. Here, we describe the encapsulation of BRP-201 into poly(lactide-co-glycolide) (PLGA) and ethoxy acetalated dextran (Ace-DEX) nanoparticles (NPs), aiming to overcome these detrimental pharmacokinetic limitations and to enhance the bioactivity of BRP-201. NPs loaded with BRP-201 were produced via nanoprecipitation and the physicochemical properties of the NPs were analyzed in-depth using dynamic light scattering (size, dispersity, degradation), electrophoretic light scattering (effective charge), NP tracking analysis (size, dispersity), scanning electron microscopy (size and morphology), UV–VIS spectroscopy (drug loading), an analytical ultracentrifuge (drug release, degradation kinetics), and Raman spectroscopy (chemical attributes). Biological assays were performed to study cytotoxicity, cellular uptake, and efficiency of BRP-201-loaded NPs versus free BRP-201 to suppress leukotriene formation in primary human leukocytes and whole blood. Both PLGA- and Ace-DEX-based NPs were significantly more efficient to inhibit leukotriene formation in neutrophils versus free drug. Whole blood experiments revealed that encapsulation of BRP-201 into Ace-DEX NPs strongly increases its potency, especially upon pro-longed (≥ 5 h) incubations and upon lipopolysaccharide-challenge of blood. Finally, intravenous injection of BRP-201-loaded NPs significantly suppressed leukotriene levels in blood of mice in vivo. These results reveal the feasibility of our pharmacological approach using a novel FLAP antagonist encapsulated into Ace-DEX-based NPs with improved efficiency in blood to suppress leukotriene biosynthesis.

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Isolation and Synthesis of a Hemoregulatory Peptide

Zeitschrift für Naturforschung C ◽

10.1515/znc-1982-11-1236 ◽

1982 ◽

Vol 37 (11-12) ◽

pp. 1297-1300 ◽

Cited By ~ 57

Author(s):

Walter R. Paukovits ◽

Ole D. Laerum

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Pure Form ◽

Structural Investigations ◽

Human Leukocytes ◽

Hemopoietic System

Abstract A peptide was isolated in pure form from human leukocytes which strongly inhibits the proliferation of immature meyloid cells in vitro (committed stem cells). Structural investigations yielded pGlu-Asp or Glu-Asp or Glu-Cys-Lys-OH as the probable sequence of this peptide. The Glu2,Asp3-analog, prepared synthetically, displayed similar activities and when applied in vivo showed effects on the hemopoietic system ranging from an inhibition of pluripotent and committed stem cells to variations in the bone marrow proliferation and alterations in peripheral blood counts.

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603 Covalent attachment of a TLR7/8 agonist to tumor-targeting antibodies drives potent anti-tumor efficacy by synergistically activating FcgR- and TLR- signaling and enables safe systemic administration

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0603 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A638-A638

Author(s):

Shelley Ackerman ◽

Felix Hartmann ◽

Cecelia Pearson ◽

Joseph Gonzalez ◽

Po Yi Ho ◽

...

Keyword(s):

Dendritic Cells ◽

Tumor Targeting ◽

Systemic Administration ◽

Covalent Attachment ◽

Preclinical Models ◽

Tlr Signaling ◽

Human Leukocytes ◽

Tlr Agonists ◽

Antigen Presenting

BackgroundImmune stimulating antibody conjugates (ISACs) covalently attach TLR7/8 immune stimulants to tumor-targeting antibodies. ISACs can be delivered systemically and act locally in the tumor microenvironment by requiring the following biological steps to elicit immune activation: 1) tumor antigen recognition, 2) Fc receptor mediated phagocytosis by myeloid antigen presenting cells (APCs), and 3) activation of endosomal TLR7 and TLR8. Here, we demonstrate that covalent attachment of our TLR7/8 agonist to tumor-targeting antibodies not only enables the resulting ISACs to be safely administered systemically in preclinical models, but also unexpectedly promotes synergy between the FcgR and TLR pathways that results in amplified anti-tumor immunity in mice and robust immune activation in human leukocytes as compared to the co-administration of the components.MethodsISAC activity and mechanistic studies were analyzed via flow cytometry, ELISA and CyTOF following in vitro coculture of human leukocytes with tumor cell lines. In vivo efficacy of HER2-targeting ISACs following systemic administration was assessed in a trastuzumab-resistant HER2+ human tumor xenograft model. Safety and tolerability were assessed in tumor-bearing mice and healthy non-human primates (NHP).ResultsWhile co-administration of intratumoral TLR7/8 agonist and intraperitoneal trastuzumab failed to control tumor growth, systemic administration of the same TLR7/8 agonist and trastuzumab in our ISAC format was efficacious and induced complete tumor regression in an Fc- and TLR-dependent manner. Analysis of primary human leukocytes stimulated with ISACs in tumor co-culture assays indicated that ISACs elicit amplified and sustained phosphorylation of Fc and TLR signaling pathways, such as pERK1/2 and pIRF-7, as compared to the unconjugated mixture of the same TLR7/8 agonist and tumor targeted antibody. ISAC stimulation was largely restricted to antigen presenting cells such as dendritic cells and plasmacytoid dendritic cells that express the relevant Fc receptors and TLR7 and/or TLR8. Modifications to the ISAC that reduce FcgR engagement (N297A/Q) or render the agonist inactive halted ISAC-mediated activation and in vivo anti-tumor efficacy. Lastly, our HER2-targeting ISACs were well-tolerated when delivered systemically in mice and NHPs.ConclusionsOur ISACs enable potent TLR agonists to be safely administered systemically in preclinical models. ISACs provide distinct and unexpected advantages over unconjugated TLR agonists, notably by driving synergy between FcgR and TLR pathways, leading to robust myeloid activation and anti-tumor efficacy. These data support the evaluation of BDC-1001, a HER2-targeted ISAC in the ongoing Phase 1/2 trial (NCT04278144).

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