Promyelocytic Cell Line HL-60 Research Articles

Protein Signature Differentiating Neutrophils and Myeloid-Derived Suppressor Cells Determined Using a Human Isogenic Cell Line Model and Protein Profiling.

Myeloid-derived suppressor cells (MDSCs) play an essential role in suppressing the antitumor activity of T lymphocytes in solid tumors, thus representing an attractive therapeutic target to enhance the efficacy of immunotherapy. However, the differences in protein expression between MDSCs and their physiological counterparts, particularly polymorphonuclear neutrophils (PMNs), remain inadequately characterized, making the specific identification and targeting of MDSCs difficult. PMNs and PMN-MDSCs share markers such as CD11b+CD14-CD15+/CD66b+, and some MDSC-enriched markers are emerging, such as LOX-1 and CD84. More proteomics studies are needed to identify the signature and markers for MDSCs. Recently, we reported the induced differentiation of isogenic PMNs or MDSCs (referred to as iPMNs and iMDSCs, respectively) from the human promyelocytic cell line HL60. Here, we profiled the global proteomics and membrane proteomics of these cells with quantitative mass spectrometry, which identified a 41-protein signature ("cluster 6") that was upregulated in iMDSCs compared with HL60 and iPMN. We further integrated our cell line-based proteomics data with a published proteomics dataset of normal human primary monocytes and monocyte-derived MDSCs induced by cancer-associated fibroblasts. The analysis identified a 38-protein signature that exhibits an upregulated expression pattern in MDSCs compared with normal monocytes or PMNs. These signatures may provide a hypothesis-generating platform to identify protein biomarkers that phenotypically distinguish MDSCs from their healthy counterparts, as well as potential therapeutic targets that impair MDSCs without harming normal myeloid cells.

Granulocyte pro-myeloperoxidase is redundantly processed by proprotein convertase furin and PC7 in HL-60 cells.

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.

Neutrophil-like cells derived from the HL-60 cell-line as a genetically-tractable model for neutrophil degranulation.

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity.

Dengue virus infection in mice induces bone marrow myeloid cell differentiation and generates Ly6Glow immature neutrophils with modulated functions.

While neutrophil activation during dengue virus infection is known, the effect of dengue virus infection on neutrophil biogenesis has not been studied. We demonstrate that dengue virus serotype 2 induces the differentiation of mice progenitor cells ex vivo toward the CD11b+Ly6C+Ly6G+ granulocyte population. We further observed an expansion of CD11b+Ly6CintLy6Glow myeloid cells in the bone marrow of dengue virus serotype 2-infected AG129 mice with low CXCR2 expression, implying an immature population. Additionally, dengue virus serotype 2 alone could induce the differentiation of promyelocyte cell line HL-60 into neutrophil-like cells, as evidenced by increased expression of CD10, CD66b, CD16, CD11b, and CD62L, corroborating the preferential shift toward neutrophil differentiation by dengue virus serotype 2 in the mouse model of dengue infection. The functional analysis showed that dengue virus serotype 2-induced neutrophil-like cells exhibited reduced phagocytic activity and enhanced NETosis, as evidenced by the increased production of myeloperoxidase, citrullinated histones, extracellular DNA, and superoxide. These neutrophil-like cells lose their ability to proliferate irreversibly and undergo arrest in the G0 to G1 phase of the cell cycle. Further studies show that myeloperoxidase-mediated signaling operating through the reactive oxygen species axis may be involved in dengue virus serotype 2-induced proliferation and differentiation of bone marrow cells as ABAH, a myeloperoxidase inhibitor, limits cell proliferation in vitro and ex vivo, affects the cell cycle, and reduces reactive oxygen species production. Additionally, myeloperoxidase inhibitor reduced NETosis and vascular leakage in dengue virus serotype 2-infected AG129 mice. Our study thus provides evidence that dengue virus serotype 2 can accelerate the differentiation of bone marrow progenitor cells into neutrophils through myeloperoxidase and modulate their functions.

The HL-60 human promyelocytic cell line constitutes an effective in vitro model for evaluating toxicity, oxidative stress and necrosis/apoptosis after exposure to black carbon particles and 2.45 GHz radio frequency

The cellular and molecular mechanisms by which atmospheric pollution from particulate matter and/or electromagnetic fields (EMFs) may prove harmful to human health have not been extensively researched. We analyzed whether the combined action of EMFs and black carbon (BC) particles induced cell damage and a pro-apoptotic response in the HL-60 promyelocytic cell line when exposed to 2.45 GHz radio frequency (RF) radiation in a gigahertz transverse electromagnetic (GTEM) chamber at sub-thermal specific absorption rate (SAR) levels. RF and BC induced moderately significant levels of cell damage in the first 8 or 24 h for all exposure times/doses and much greater damage after 48 h irradiation and the higher dose of BC. We observed a clear antiproliferative effect that increased with RF exposure time and BC dose. Oxidative stress or ROS production increased with time (24 or 48 h of radiation), BC dose and the combination of both. Significant differences between the proportion of damaged and healthy cells were observed in all groups. Both radiation and BC participated separately and jointly in triggering necrosis and apoptosis in a programmed way. Oxidative-antioxidant action activated mitochondrial anti-apoptotic BCL2a gene expression after 24 h irradiation and exposure to BC. After irradiation of the cells for 48 h, expression of FASR cell death receptors was activated, precipitating the onset of pro-apoptotic phenomena and expression and intracellular activity of caspase-3 in the mitochondrial pathways, all of which can lead to cell death. Our results indicate that the interaction between BC and RF modifies the immune response in the human promyelocytic cell line and that these cells had two fates mediated by different pathways: necrosis and mitochondria-caspase dependent apoptosis. The findings may be important in regard to antimicrobial, inflammatory and autoimmune responses in humans.

Expression of the phagocytic receptors αMβ2 and αXβ2 is controlled by RIAM, VASP and Vinculin in neutrophil-differentiated HL-60 cells.

Activation of the integrin phagocytic receptors CR3 (αMβ2, CD11b/CD18) and CR4 (αXβ2, CD11c/CD18) requires Rap1 activation and RIAM function. RIAM controls integrin activation by recruiting Talin to β2 subunits, enabling the Talin-Vinculin interaction, which in term bridges integrins to the actin-cytoskeleton. RIAM also recruits VASP to phagocytic cups and facilitates VASP phosphorylation and function promoting particle internalization. Using a CRISPR-Cas9 knockout approach, we have analyzed the requirement for RIAM, VASP and Vinculin expression in neutrophilic-HL-60 cells. All knockout cells displayed abolished phagocytosis that was accompanied by a significant and specific reduction in ITGAM (αM), ITGAX (αX) and ITGB2 (β2) mRNA, as revealed by RT-qPCR. RIAM, VASP and Vinculin KOs presented reduced cellular F-actin content that correlated with αM expression, as treatment with the actin filament polymerizing and stabilizing drug jasplakinolide, partially restored αM expression. In general, the expression of αX was less responsive to jasplakinolide treatment than αM, indicating that regulatory mechanisms independent of F-actin content may be involved. The Serum Response Factor (SRF) was investigated as the potential transcription factor controlling αMβ2 expression, since its coactivator MRTF-A requires actin polymerization to induce transcription. Immunofluorescent MRTF-A localization in parental cells was primarily nuclear, while in knockouts it exhibited a diffuse cytoplasmic pattern. Localization of FHL-2 (SRF corepressor) was mainly sub-membranous in parental HL-60 cells, but in knockouts the localization was disperse in the cytoplasm and the nucleus, suggesting RIAM, VASP and Vinculin are required to maintain FHL-2 close to cytoplasmic membranes, reducing its nuclear localization and inhibiting its corepressor activity. Finally, reexpression of VASP in the VASP knockout resulted in a complete reversion of the phenotype, as knock-ins restored αM expression. Taken together, our results suggest that RIAM, VASP and Vinculin, are necessary for the correct expression of αMβ2 and αXβ2 during neutrophilic differentiation in the human promyelocytic HL-60 cell line, and strongly point to an involvement of these proteins in the acquisition of a phagocytic phenotype.

Altered sialidase expression in human myeloid cells undergoing apoptosis and differentiation

To gain insight into sialic acid biology and sialidase/neuraminidase (NEU) expression in mature human neutrophil (PMN)s, we studied NEU activity and expression in PMNs and the HL60 promyelocytic leukemic cell line, and changes that might occur in PMNs undergoing apoptosis and HL60 cells during their differentiation into PMN-like cells. Mature human PMNs contained NEU activity and expressed NEU2, but not NEU1, the NEU1 chaperone, protective protein/cathepsin A(PPCA), NEU3, and NEU4 proteins. In proapoptotic PMNs, NEU2 protein expression increased > 30.0-fold. Granulocyte colony-stimulating factor protected against NEU2 protein upregulation, PMN surface desialylation and apoptosis. In response to 3 distinct differentiating agents, dimethylformamide, dimethylsulfoxide, and retinoic acid, total NEU activity in differentiated HL60 (dHL60) cells was dramatically reduced compared to that of nondifferentiated cells. With differentiation, NEU1 protein levels decreased > 85%, PPCA and NEU2 proteins increased > 12.0-fold, and 3.0-fold, respectively, NEU3 remained unchanged, and NEU4 increased 1.7-fold by day 3, and then returned to baseline. In dHL60 cells, lectin blotting revealed decreased α2,3-linked and increased α2,6-linked sialylation. dHL60 cells displayed increased adhesion to and migration across human bone marrow-derived endothelium and increased bacterial phagocytosis. Therefore, myeloid apoptosis and differentiation provoke changes in NEU catalytic activity and protein expression, surface sialylation, and functional responsiveness.

N-Succinyl-S-Farnesyl-L-Cysteine (SFC): A Novel Isoprenylcysteine Analog with In Vitro Anti-Inflammatory Activity and Clinical Skin Protecting Properties

Over the past 15 years, small molecule isoprenylcysteine (IPC) analogs have been identified as a potential new class of topical anti-inflammatories. Clinical studies have demonstrated that IPCs are both safe and effective in promoting healthy skin when applied topically. This work aims to demonstrate N-Succinyl-S-farnesyl-L-cysteine (SFC) as a novel IPC molecule that provides a broad spectrum of benefits for skin. Human promyelocytic cell line HL-60, human dermal microvascular endothelial cells (HDMECs), human dermal fibroblasts (HDFs), and normal human epidermal keratinocytes (NHEKs) were exposed in culture to various inducers to trigger reactive oxygen species, cytokines, or collagenase production. A 49-subject randomized double-blind, vehicle-controlled, split face trial was performed with 1% SFC gel, or 5% niacinamide and vehicle applied for 12 weeks to evaluate anti-wrinkle and anti-aging endpoints. We demonstrated that SFC inhibited GPCR and TLR-induced pro-inflammatory cytokine release in NHEKs and HDMECs from several inflammatory inducers such as UVB, chemicals, cathelicidin, and bacteria. SFC successfully reduced GPCR-induced oxidation in differentiated neutrophils. Moreover, photoaging studies showed that SFC reduced UVA-induced collagenase (pro-MMP-1) production in HDFs. Clinical assessment of 1% SFC gel demonstrated improvement above the vehicle for wrinkle reduction, hydration, texture, and overall appearance of skin. N-Succinyl-S-farnesyl-L-cysteine (SFC) is a novel anti-inflammatory small molecule and is the first farnesyl-cysteine IPC shown to clinically improve appearance and signs of aging, while also having the potential to ameliorate inflammatory skin disorders.

Antiproliferative activity of extract from in vitro callus cultures of Astragalus vesicarius ssp. carniolicus (A. Kern.) Chater

Five isoflavonoids, i.e. 5-hydroxy-7-methoxy-2’, 5’-dihydroxyisoflavone (AV4), 5, 7-dihydroxy-4’-methoxyisoflavone (AV6), 7-methoxy-5-hydroxy-4’-methoxy-2’-hydroxyisoflavone (AV7), 8-pregnyl genistein (AV9), 5,7-dihydroxy-8-pregnyl-4’-methoxy-2’-hydroxyisoflavone (AV10) and one coumarochromone – sophorophenolone (AV8) were isolated from EtOAc of in vitro callus cultures of Astragalus vesicarius ssp. carniolicus, after enzymatic hydrolysis with β-glucosidase. Their structures were tentatively elucidated by spectroscopic mean (1H NMR and HR-ESI-MS spectra). Antiproliferative activity of EtOAc extract and isolated aglycones against chemosensitive human promyelocyte cell line HL-60 and its multidrug-resistant variant HL-60/Dox was assessed in vitro. Despite the strong activity of EtOAc (IC50 8.8 µg/mL (HL-6, 72 h) to 11.8 µg/mL (HL-60/Dox, 72 h)), prenylated compound AV9 showed also antiproliferative activity – 36.1 µg/mL (HL-60 and HL-60/Dox, 72 h).

Human Isogenic Cell Line Models for Neutrophils and Myeloid-Derived Suppressor Cells

Neutrophils with immunosuppressive activity are polymorphonuclear myeloid-derived suppressor cells (MDSCs) and may contribute to the resistance to cancer immunotherapy. A major gap for understanding and targeting these cells is the paucity of cell line models with cardinal features of human immunosuppressive neutrophils and their normal counterparts, especially in an isogenic manner. To address this issue, we employ the human promyelocytic cell line HL60 and use DMSO and cytokines (granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 6 (IL6)) to induce the formation of either neutrophils or MDSCs. The induced MDSCs are CD11b+ CD33+ HLA-DR−/low and are heterogeneous for CD15 and CD14 expression. The induced MDSCs abrogate IL2 production and activation-induced cell death of the human T cell line Jurkat stimulated by CD3/CD28 antibodies, whereas the induced neutrophils enhance IL2 production from Jurkat cells. The induced MDSCs upregulate the expression of C/EBPβ, STAT3, VEGFR1, FATP2 and S100A8. Lastly, the immunosuppressive activity of the induced MDSCs is inhibited by all-trans retinoic acid and STAT3 inhibitor BP-1-102 through cellular differentiation and dedifferentiation mechanisms, respectively. Together, our study establishes a human isogenic cell line system for neutrophils and MDSCs and this system is expected to facilitate future studies on the biology and therapeutics of human immunosuppressive neutrophils.

RIAM-VASP Module Relays Integrin Complement Receptors in Outside-In Signaling Driving Particle Engulfment.

The phagocytic integrins and complement receptors αMβ2/CR3 and αXβ2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of β2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

Mutation-Specific Dominant Negative Effects Determine the Phenotype in SRP54 Deficiency

Whole exome sequencing analyses are increasingly performed on patients presenting with suspected inherited disease but lacking classical mutations linked to presented phenotypes. Using whole-exome sequencing in SBDS-negative Shwachman-Diamond Syndrome (SDS) families, we recently identified three independent patients, each of whom carried a heterozygous de novo missense variant of SRP54 (encoding signal recognition particle 54 kDa). The SRP54 protein is a key component of the ribonucleoprotein complex that mediates the co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum (ER). Whilst two of the identified patients were carrying nucleotide transversion in SRP54 (p.T115A and p.G226E), which manifested in typical SDS features like neutropenia and exocrine pancreatic insufficiency, the third patient was carrying a nucleotide deletion (p.T117Δ), which only manifested in mild neutropenia without additional SDS features (Carapito et al. 2017, JCI). Here, we describe a zebrafish knock-out (KO) mutant as the very first transgenic in vivo model of SRP54 deficiency, translate our previous findings into living organisms and propose disease-driving mechanisms. We show that homozygous srp54 mutant zebrafish are suffering not only from severe neutropenia as shown by flow cytometry and Whole-Mount-In-Situ Hybridization (WISH), but also from gross developmental defects leading to early embryonic lethality. In fact, srp54-/- zebrafish did not survive more than 72 hours post fertilization, indicating that complete loss of Srp54 is not compatible with life. Injection with wild-type human SRP54 mRNA induced transient restoration of SRP54 protein expression and slightly enhanced the survival of the homozygous mutants. However, long-term viability could not be restored, revealing that srp54 is not only critically required during early embryogenesis but also at later stages of development. Heterozygous siblings on the other hand are viable and display only mild neutropenia but no pancreas defects. Interestingly however, injection of mutant mRNAs of human SRP54 (p.T115A, p.T117Δ, p.226E) into heterozygous srp54 KO mutants aggravated the phenotype inducing more profound neutropenia and pancreas changes similar to those observed in classical SDS patients. Of note, these effects were more severe for the transversions p.T115A and p.G226E. Mutation p.T117Δ only caused a minor reduction in the number of neutrophils, without affecting the pancreas. To further investigate SRP54 driven neutrophil defects, we used lentiviral transduction to exogenously express human SRP54 mutant variants in promyelocytic HL-60 cells. When stimulating these cells to differentiate by ATRA treatment, we found significantly impaired morphologic differentiation and CD11b surface induction compared to control cells. The severity of these effects was again specific to the three different identified mutations, with p.T115A and p.G226E being more severe than p.T117Δ. These findings confirm the type-specific effects of SRP54 mutations and indicate that SRP54 defects interfere with neutrophil differentiation and thus ultimately lead to neutropenia. Collectively, we here describe a novel zebrafish disease model of SDS and congenital neutropenia founding on SRP54 as molecular driver. Our model demonstrates that at least one healthy allele of srp54 is pivotal for survival, which is in line with the findings in humans, where homozygous mutations in SRP54 have never been detected. We reveal that the phenotypic manifestation of heterozygous SRP54 mutations strongly depends on the type of mutation: while mutations likely causing a simple SRP54 loss of function (e.g. p.T117Δ) induce a rather mild phenotype characterized by moderate neutropenia only (analogous to the heterozygous fish mutant), more severe SDS-like phenotypes involve SRP54 mutations that exert dominant negative effects (e.g. p.T115A and p.G226E). Ultimately, we make use of the promyelocytic cell line HL-60 to propose neutrophil differentiation defects as the underlying cause of SRP54 driven neutropenia. At the time being, RNA sequencing and protein expression analyses are performed in our laboratory, which will add to the understanding of the mechanistical background of the neutrophilic differentiation blockage and eventually uncover novel treatment strategies for SRP54 deficiency. Disclosures No relevant conflicts of interest to declare.

Isolation of cytotoxic monomeric protein and morin derivatives from Solena amplexicaulis (Lam.) Gandhi

Solena amplexicaulis (Lam.) Gandhi (family- Cucurbitaceae), is used both in the Indian traditional system and folk medicine to treat several pathophysiological conditions and complex diseases including cancer. The screening of the phytochemicals of this plant (aerial parts) was performed to evaluate their cytotoxic effect against an in vitro cancer model utilising acute promyelocytic leukaemia HL60 cell line. Phytoconstituents were isolated by column chromatography and characterised. The purified protein was extracted, isolated and purified by using standard techniques. The cytotoxicity was evaluated by MTT assay. Spectral analysis revealed the isolated phytochemicals to be Morin-3-O-xyloside (1) and Morin 3-O-glucoside (2). The purified protein (P1) was found to be monomeric having a molecular weight of 30.2 kDa. Watching over 24 h exposure, compound 1 (IC50 1.5 µmol/L), compound 2 (IC50 3.5 µmol/L), and P1 (2.67 µmol/L) exhibited significant cytotoxic activity.

Antitumour effects of selected plant polyphenols, gallic acid and ellagic acid, on sensitive and multidrug-resistant leukaemia HL60 cells.

The aim of this study was to examine the antitumour effects of plant phenolic acids, gallic acid (GA) and ellagic acid (EA), on human promyelocytic leukaemia sensitive HL60 cell line and its resistant sublines exhibiting two MDR phenotypes: HL60/VINC (overexpressing P-glycoprotein) and HL60/MX2 (characterized by the presence of mutated α isoform of topoisomerase II). Both studied compounds exerted comparable cytotoxic activities towards sensitive HL60 cells and their MDR counterparts. It was also found that GA and EA modulated the cellular level of reactive oxygen species in a dose-dependent and time-dependent manner. Furthermore, it was demonstrated that GA (IC90 ) and EA (IC50 and IC90 ) significantly increased the percentage of sub-G1 subpopulation of all studied leukaemia cells causing oligonucleosomal DNA fragmentation. Both compounds used at IC90 triggered mainly the apoptotic death of these cells. However, GA had no effect on the activity of caspase-3 as well as caspase-8 in sensitive HL60 cells and their MDR counterparts. In contrast, EA provoked a significant activation of these caspases in all studied leukaemia cells. It was also found that lysosomes were not involved in triggering programmed death of sensitive HL60 and MDR cells by GA and EA.

CD40 ligand deficiency causes functional defects of peripheral neutrophils that are improved by exogenous IFN-γ

Patients with X-linked hyper-IgM syndrome caused by CD40 ligand (CD40L) deficiency often present with episodic, cyclic, or chronic neutropenia, suggesting abnormal neutrophil development in the absence of CD40L-CD40 interaction. However, even when not neutropenic and despite immunoglobulin replacement therapy, CD40L-deficient patients are susceptible to life-threatening infections caused by opportunistic pathogens, suggesting impaired phagocyte function and the need for novel therapeutic approaches. We sought to analyze whether peripheral neutrophils from CD40L-deficient patients display functional defects and to explore the invitro effects of recombinant human IFN-γ (rhIFN-γ) on neutrophil function. We investigated the microbicidal activity, respiratory burst, and transcriptome profile of neutrophils from CD40L-deficient patients. In addition, we evaluated whether the lack of CD40L in mice also affects neutrophil function. Neutrophils from CD40L-deficient patients exhibited defective respiratory burst and microbicidal activity, which were improved invitro by rhIFN-γ but not soluble CD40L. Moreover, neutrophils from patients showed reduced CD16 protein expression and a dysregulated transcriptome suggestive of impaired differentiation. Similar to CD40L-deficient patients, CD40L knockout mice were found to have impaired neutrophil responses. In parallel, we demonstrated that soluble CD40L induces the promyelocytic cell line HL-60 to proliferate and mature by regulating the expression of genes of the same Gene Ontology categories (eg, cell differentiation) when compared with those dysregulated in peripheral blood neutrophils from CD40L-deficient patients. Our data suggest a nonredundant role of CD40L-CD40 interaction in neutrophil development and function that could be improved invitro by rhIFN-γ, indicating a potential novel therapeutic application for this cytokine.

Cytotoxic lanostane triterpenoids from the fruiting bodies of Piptoporus betulinus

Chemical investigation of a bioactive methanolic extract of the fruiting bodies of Piptoporus betulinus led to the isolation of five previously undescribed lanostane triterpenoids named piptolinic acids A−E, as well as five known lanostane triterpenoids. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic and HRESIMS analysis. Piptolinic acid A with an unusual moiety (3-hydroxy-4-methoxycarbonyl-3-methylbutyryloxy) at C-3 exhibited comparable cytotoxic activity against human promyelocytic leukemia cell line HL-60 (IC50 = 1.77 μM) and human acute monocytic leukemia cell line THP-1 (IC50 = 8.21 μM) to those of positive control, fluorouracil (IC50 = 6.38 and 4.41 μM, respectively).

In vivo and in vitro evaluation for nutraceutical purposes of capsaicin, capsanthin, lutein and four pepper varieties

The purpose of this study is to determine the nutraceutic potential of different Capsicum sp, capsaicin, capsanthin and lutein and provide data in order to clarify the conflicting results obtained for capsaicin by different authors. To achieve these objectives, in vivo (geno/antigenotoxicity and lifespan assays in the animal model Drosophila) and in vitro (cytotoxicity and DNA-fragmentation assays in HL60 promyelocytic cell line) assays were carried out. Results showed that i) none of the tested substances were genotoxic except green hot pepper and capsaicin at the highest tested concentration (5 mg/mL and 11.5 μM respectively), ii) all tested substances except green hot pepper are antimutagenic against H2O2-induced damage, iii) only red sweet pepper significantly extend the lifespan and healthspan of D. melanogaster at 1.25 and 2.5 mg/mL, iv) all pepper varieties induce dose-depended cytotoxic effect in HL60 cells with different IC50, and v) all pepper varieties and capsaicin exerted proapoptotic effect on HL60 cells. In conclusion: (i) sweet peppers could be suggested as nutraceutical food, (ii) hot peppers should be moderately consumed, and (iii) supplementary studies are necessary to clarify the synergic effect of the carotenoids and capsaicinoids in the hot pepper food matrix.

High-throughput downstream process development for cell-based products using aqueous two-phase systems

As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research.

Polyhalogenated Indoles from the Red Alga Rhodophyllis membranacea: The First Isolation of Bromo-Chloro-Iodo Secondary Metabolites.

An unusual tetrahalogenated indole with the exceptionally rare inclusion of the three halogens bromine, chlorine, and iodine was found using mass spectrometry within a fraction of a semipurified extract obtained from the red alga Rhodophyllis membranacea. We report herein the isolation and structure elucidation, using a combination of NMR spectroscopy and mass spectrometry, of 11 new tetrahalogenated indoles (1-11), including four bromochloroiodoindoles (5-7, 10). Several were evaluated for cytotoxic and antifungal activities against the HL-60 promyelocytic cell line and Saccharomyces cerevisiae, respectively.

Generation of Adducts of 4-Hydroxy-2-nonenal with Heat Shock 60 kDa Protein 1 in Human Promyelocytic HL-60 and Monocytic THP-1 Cell Lines.

Heat shock 60 kDa protein 1 (HSP60) is a chaperone and stress response protein responsible for protein folding and delivery of endogenous peptides to antigen-presenting cells and also a target of autoimmunity implicated in the pathogenesis of atherosclerosis. By two-dimensional electrophoresis and mass spectrometry, we found that exposure of human promyelocytic HL-60 cells to a nontoxic concentration (10 μM) of 4-hydroxy-2-nonenal (HNE) yielded a HSP60 modified with HNE. We also detected adducts of HNE with putative uncharacterized protein CXorf49, the product of an open reading frame identified in various cell and tissue proteomes. Moreover, exposure of human monocytic THP-1 cells differentiated with phorbol 12-myristate 13-acetate to 10 μM HNE, and to light density lipoprotein modified with HNE (HNE-LDL) or by copper-catalyzed oxidation (oxLDL), but not to native LDL, stimulated the formation of HNE adducts with HSP60, as detected by immunoprecipitation and western blot, well over basal levels. The identification of HNE-HSP60 adducts outlines a framework of mutually reinforcing interactions between endothelial cell stressors, like oxLDL and HSP60, whose possible outcomes, such as the amplification of endothelial dysfunction, the spreading of lipoxidative damage to other proteins, such as CXorf49, the activation of antigen-presenting cells, and the breaking of tolerance to HSP60 are discussed.