Antiviral Immunoglobulins of Chicken Egg Yolk for Potential Prevention of SARS-CoV-2 Infection.

Background: Some viruses cause outbreaks, which require immediate attention. Neutralizing antibodies could be developed for viral outbreak management. However, the development of monoclonal antibodies is often long, laborious, and unprofitable. Here, we report the development of chicken polyclonal neutralizing antibodies against SARS-CoV-2 infection. Methods: Layers were immunized twice with 14-day intervals using the purified receptor-binding domain (RBD) of the S protein of SARS-CoV-2/Wuhan or SARS-CoV-2/Omicron. Eggs were harvested 14 days after the second immunization. Polyclonal IgY antibodies were extracted. Binding of anti-RBD IgYs was analyzed by immunoblot and indirect ELISA. Furthermore, the neutralization capacity of anti-RBD IgYs was measured in Vero-E6 cells infected with SARS-CoV-2-mCherry/Wuhan and SARS-CoV-2/Omicron using fluorescence and/or cell viability assays. In addition, the effect of IgYs on the expression of SARS-CoV-2 and host cytokine genes in the lungs of Syrian Golden hamsters was examined using qRT-PCR. Results: Anti-RBD IgYs efficiently bound viral RBDs in situ, neutralized the virus variants in vitro, and lowered viral RNA amplification, with minimal alteration of virus-mediated immune gene expression in vivo. Conclusions: Altogether, our results indicate that chicken polyclonal IgYs can be attractive targets for further pre-clinical and clinical development for the rapid management of outbreaks of emerging and re-emerging viruses.

Identifying Lysophosphatidic Acid Acyltransferaseâ€ ß (LPAAT-ß) as the Target of a Nanomolar Angiogenesis Inhibitor from a Phenotypic Screen Using the Polypharmacology Browser PPB2.

By screening a focused library of kinase inhibitor analogues in a phenotypic co-culture assay for angiogenesis inhibition, we identified an aminotriazine that acts as a cytostatic nanomolar inhibitor. However, this aminotriazine was found to be completely inactive in a whole-kinome profiling assay. To decipher its mechanism of action, we used the online target prediction tool PPB2 (http://ppb2.gdb.tools), which suggested lysophosphatidic acid acyltransferaseâ€…ß (LPAAT-ß) as a possible target for this aminotriazine as well as several analogues identified by structure-activity relationship profiling. LPAAT-ß inhibition (IC50 ≈15 nm) was confirmed in a biochemical assay and by its effects on cell proliferation in comparison with a known LPAAT-ß inhibitor. These experiments illustrate the value of target-prediction tools to guide target identification for phenotypic screening hits and significantly expand the rather limited pharmacology of LPAAT-ß inhibitors.

Optical micromanipulation of nanoparticles and cells inside living zebrafish.

Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology.

Zebrafish as a model system for characterization of nanoparticles against cancer.

Therapeutic nanoparticles (NPs) have great potential to deliver drugs against human diseases. Encapsulation of drugs in NPs protects them from being metabolized, while they are delivered specifically to a target site, thereby reducing toxicity and other side-effects. However, non-specific tissue accumulation of NPs, for example in macrophages, especially in the spleen and liver is a general problem with many NPs being developed for cancer therapy. To address the problem of non-specific tissue accumulation of NPs we describe the development of the zebrafish embryo as a transparent vertebrate system for characterization of NPs against cancer. We show that injection of human cancer cells results in tumor-like structures, and that subsequently injected fluorescent NPs, either made of polystyrene or liposomes can be imaged in real-time. NP biodistribution and general in vivo properties can be easily monitored in embryos having selective fluorescent labeling of specific tissues. We demonstrate in vitro, by using optical tweezer micromanipulation, microscopy and flow cytometry that polyethylene glycol (PEG) coating of NPs decreases the level of adhesion of NPs to macrophages, and also to cancer cells. In vivo in zebrafish embryos, PEG coating resulted in longer NP circulation times, decreased macrophage uptake, and reduced adhesion to the endothelium. Importantly, liposomes were observed to accumulate passively and selectively in tumor-like structures comprised of human cancer cells. These results show that zebrafish embryo is a powerful system for microscopy-based screening of NPs on the route to preclinical testing.

In Vitro Characterization of Valproic Acid, ATRA, and Cytarabine Used for Disease-Stabilization in Human Acute Myeloid Leukemia: Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells.

The combined use of the histone deacetylase inhibitor valproic acid (VPA), the retinoic acid receptor- α agonist all-trans retinoic acid (ATRA), and the deoxyribonucleic acid polymerase- α inhibitor cytarabine (Ara-C) is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML). Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

Tumor versus stromal cells in culture--survival of the fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

Contextual compound screening for improved therapeutic discovery.

Cellular behaviors are governed by combinations of systemic and microenvironmental factors; together, these regulate cell signaling responses to growth factors. This contextual microenvironmental influence also determines drug sensitivity. Hence using in vitro systems that model contextual cellular behavior is highly beneficial for effective therapeutic development. Angiogenesis (formation of blood vessels) is driven by a series of dynamic endothelial cell signaling responses to growth factors under the influence of the vascular extracellular matrix and adjacent pericytes. In vitro primary human vascular cell co-cultures self-assemble into capillary-like structures through reciprocal heterotypic interactions that mimic angiogenic context dynamics. By using temporal live-cell imaging-based analysis, unique angiogenic microenvironments can be delineated to quantify the contextual activity of compound inhibitors. We used this in vitro organotypic contextual screening approach to conduct structure-activity relationship analysis on a combretastatin A-4 analogue series to identify novel compounds with potent vascular disrupting activity in vivo.

Domains I and IV of annexin A2 affect the formation and integrity of in vitro capillary-like networks.

Annexin A2 (AnxA2) is a widely expressed multifunctional protein found in different cellular compartments. In spite of lacking a hydrophobic signal peptide, AnxA2 is found at the cell surface of endothelial cells, indicative of a role in angiogenesis. Increased extracellular levels of AnxA2 in tumours correlate with neoangiogenesis, metastasis and poor prognosis. We hypothesised that extracellular AnxA2 may contribute to angiogenesis by affecting endothelial cell-cell interactions and motility. To address this question, we studied the effect of heterotetrameric and monomeric forms of AnxA2, as well as its two soluble domains on the formation and maintenance of capillary-like structures by using an in vitro co-culture system consisting of endothelial and smooth muscle cells. In particular, addition of purified domains I and IV of AnxA2 potently inhibited the vascular endothelial growth factor (VEGF)-dependent formation of the capillary-like networks in a dose-dependent manner. In addition, these AnxA2 domains disrupted endothelial cell-cell contacts in preformed capillary-like networks, resulting in the internalisation of vascular endothelial (VE)-cadherin and the formation of VE-cadherin-containing filopodia-like structures between the endothelial cells, suggesting increased cell motility. Addition of monoclonal AnxA2 antibodies, in particular against Tyr23 phosphorylated AnxA2, also strongly inhibited network formation in the co-culture system. These results suggest that extracellular AnxA2, most likely in its Tyr phosphorylated form, plays a pivotal role in angiogenesis. The exogenously added AnxA2 domains most likely mediate their effects by competing with endogenous AnxA2 for extracellular factors necessary for the initiation and maintenance of angiogenesis, such as those involved in the formation/integrity of cell-cell contacts.

Image-based high-throughput screening for inhibitors of angiogenesis.

Automated multicolor fluorescence microscopy facilitates high-throughput quantitation of cellular parameters of complex, organotypic systems. In vitro co-cultured vascular cells form capillary-like networks that model facets of angiogenesis, making it an attractive alternative for anti-angiogenic drug discovery. We have adapted this angiogenesis assay system to a high-throughput format to enable automated image-based high-throughput screening of live primary human vascular cell co-cultures with chemical libraries for anti-angiogenic drug discovery. Protocols are described for setup of a fluorescence-based co-culture assay, live cell image acquisition, image analysis of morphological parameters, and screening data handling.

Camptothecin-7-yl-methanthiole: semisynthesis and biological evaluation.

The introduction of a methylenthiol group at position 7 of camptothecin was carried out in four steps. This preparation also yielded the corresponding disulfide, which behaves as a prodrug due to its reactivity with glutathione. Assessment of their antiproliferative activities, investigations of their mechanism of action, and molecular modeling analysis indicated that the 7-modified camptothecin derivatives described herein maintain the biological activity and drug-target interactions of the parent compound.

Soluble mediators released by acute myeloid leukemia cells increase capillary-like networks.

Increased bone marrow angiogenesis is seen in several hematological malignancies, including acute myeloid leukemia (AML). We used a co-culture assay of endothelial and vascular smooth muscle cells (vSMC) to investigate the effects of AML-conditioned medium on capillary networks. We investigated primary AML cells derived from 44 unselected patients and observed that for a large subset of patients, the constitutive cytokine release by the leukemic cells stimulated endothelial cell organization into capillary-like networks, while there were only minor or no effects for other patients. We analyzed the constitutive AML cell release of 31 cytokines for all the patients and performed a hierarchical cluster analysis of the cytokine profile which identified two major patient subsets that differed in their ability to enhance capillary-like networks; increased capillary-like networks was then associated with high constitutive release of several cytokines and especially high levels of several pro-angiogenic chemokines. Significantly increased network formation was not seen for any of the 11 acute lymphoblastic leukemia patients investigated. The cytokine response by activated normal T cells inhibited endothelial network formation in our in vitro model of angiogenesis and activated normal monocytes had only a minor influence on tube formation. Our study shows that AML-derived cytokines can induce the organization of endothelial cells into vessel-like structures.

A combined targeted/phenotypic approach for the identification of new antiangiogenics agents active on a zebrafish model: from in silico screening to cyclodextrin formulation.

A combined targeted/phenotypic approach for the rapid identification of novel antiangiogenics with in vivo efficacy is herein reported. Considering the important role played by the tyrosine kinase c-Src in the regulation of tumour angiogenesis, we submitted our in-house library of c-Src inhibitors to a sequential screening approach: in silico screening on VEGFR2, in vitro screening on HUVEC cells, ADME profiling, formulation and in vivo testing on a zebrafish model. A promising antiangiogenic candidate able to interfere with the vascular growth of a zebrafish model at low micromolar concentration was thus identified.

Isosteric replacement of the Z-enone with haloethyl ketone and E-enone in a resorcylic acid lactone series and biological evaluation.

The synthesis of a small library of resorcylic acid lactones and evaluation of their biological properties as kinase inhibitors is described. Within the series E-enones were found more active than corresponding Z-enones as inhibitors of a subset of kinases containing a conserved cysteine. Replacement of the enone moiety with a ß-haloketone group led to compounds with an interesting kinase selectivity profile and also antiproliferative activity against Jurkat cells. An E-enone derivative also showed activity against capillary tube formation based on a co-culture of primary human umbilical cord endothelial cells (HUVECs) and vascular smooth muscle cells (vSMCs).

Imaged-based high-throughput screening for anti-angiogenic drug discovery.

Recent developments in high-content screening (HCS) technologies make it an attractive alternative for anti-angiogenic drug discovery. HCS integrates high-throughput methodologies with automated multicolor fluorescence microscopy to collect quantitative morphological and molecular data from complex biological systems. Organotypic systems based on primary vascular cells model many facets of angiogenesis. The adaptation of these complex in vitro assay systems to high-throughput HCS formats with automated image acquisition enables large-scale chemical library screening campaigns. These HCS principles can be extended further to allow small molecule compounds in in vivo model organisms such as zebrafish. In this review we discuss the latest developments within automated image-based high-throughput screening of chemical libraries for anti-angiogenic compounds.

Synthesis and biological evaluation of new camptothecin derivatives obtained by modification of position 20.

The preparation and biological evaluation of a novel series of dimeric camptothecin derivatives are described. All the new compounds showed a significant ability to inhibit human tumor cell growth with IC(50) values ranging from 0.03 to 12.2 µM. The interference with the activity of the nuclear enzymes topoisomerases has been demonstrated, highlighting the poison effect of one of the obtained byproducts toward topoisomerase I. A moderate antiangiogenic activity has been demonstrated for one of the obtained compounds. Moreover, the effects of four new compounds on caspases activity and ROS generation have been studied on transgenic mouse cell.

A novel imaging-based high-throughput screening approach to anti-angiogenic drug discovery.

The successful progression to the clinic of angiogenesis inhibitors for cancer treatment has spurred interest in developing new classes of anti-angiogenic compounds. The resulting surge in available candidate therapeutics highlights the need for robust, high-throughput angiogenesis screening systems that adequately capture the complexity of new vessel formation while providing quantitative evaluation of the potency of these agents. Available in vitro angiogenesis assays are either cumbersome, impeding adaptation to high-throughput screening formats, or inadequately model the complex multistep process of new vessel formation. We therefore developed an organotypic endothelial-mural cell co-culture assay system that reflects several facets of angiogenesis while remaining compatible with high-throughput/high-content image screening. Co-culture of primary human endothelial cells (EC) and vascular smooth muscle cells (vSMC) results in assembly of a network of tubular endothelial structures enveloped with vascular basement membrane proteins, thus, comprising the three main components of blood vessels. Initially, EC are dependent on vSMC-derived VEGF and sensitive to clinical anti-angiogenic therapeutics. A subsequent phenotypic VEGF-switch renders EC networks resistant to anti-VEGF therapeutics, demarcating a mature vascular phenotype. Conversely, mature EC networks remain sensitive to vascular disrupting agents. Therefore, candidate anti-angiogenic compounds can be interrogated for their relative potency on immature and mature networks and classified as either vascular normalizing or vascular disrupting agents. Here, we demonstrate that the EC-vSMC co-culture assay represents a robust high-content imaging high-throughput screening system for identification of novel anti-angiogenic agents. A pilot high-throughput screening campaign was used to define informative imaging parameters and develop a follow-up dose-response scheme for hit characterization. High-throughput screening using the EC-vSMC co-culture assay establishes a new platform to screen for novel anti-angiogenic compounds for cancer therapy.

Mural cell associated VEGF is required for organotypic vessel formation.

BACKGROUND: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. METHODS AND FINDINGS: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. CONCLUSIONS: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation.

The mRNA-binding site of annexin A2 resides in helices C-D of its domain IV.

Annexin A2 (AnxA2) is a Ca(2+)-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific approximately 100 nucleotide region in the 3'-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3'-untranslated region of anxA2 mRNA is Ca(2+)-dependent. By contrast, the interaction is Ca(2+)-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca(2+)-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.

Engineering, biophysical characterisation and binding properties of a soluble mutant form of annexin A2 domain IV that adopts a partially folded conformation.

The four approximately 75-residue domains (repeats) that constitute the annexin core structure all possess an identical five-alpha-helix bundle topology, but the physico-chemical properties of the isolated domains are different. Domain IV of the annexins has previously been expressed only as inclusion bodies, resistant to solubilisation. Analysis of the conserved, exposed hydrophobic residues of the four annexin domains reveals that domain IV contains the largest number of hydrophobic residues involved in interfacial contacts with the other domains. We designed five constructs of domain IV of annexin A2 in which several interfacial hydrophobic residues were substituted by hydrophilic residues. The mutant domain, in which all fully exposed hydrophobic interfacial residues were substituted, was isolated as a soluble protein. Circular dichroism measurements indicate that it harbours a high content of alpha-helical secondary structure and some tertiary structure. The CD-monitored (lambda=222 nm) thermal melting profile suggests a weak cooperative transition. Nuclear magnetic resonance (1H-15N) correlation spectroscopy reveals heterogeneous line broadening and an intermediate spectral dispersion. These properties are indicative of a partially folded protein in which some residues are in a fairly structured conformation, whereas others are in an unfolded state. This conclusion is corroborated by 1-anilinonaphthalene-8-sulfonate fluorescence (ANS) analyses. Surface plasmon resonance measurements also indicate that this domain binds heparin, a known ligand of domain IV in the full-length annexin A2, although with lower affinity.