extfieldsofstudy: Medicine Biology

text:
Design and evaluation of bi- and trispecific antibodies targeting multiple filovirus glycoproteins

Filoviruses (family Filoviridae) include five ebolaviruses and Marburg virus. These pathogens cause a rapidly progressing and severe viral disease with high mortality rates (generally 30–90%). Outbreaks of filovirus disease are sporadic and, until recently, were limited to less than 500 cases. However, the 2013–2016 epidemic in western Africa, caused by Ebola virus (EBOV), illustrated the potential of filovirus outbreaks to escalate to a much larger scale (over 28,000 suspected cases). mAbs against the envelope glycoprotein represent a promising therapeutic platform for managing filovirus infections. However, mAbs that exhibit neutralization or protective properties against multiple filoviruses are rare. Here we examined a panel of engineered bi- and trispecific antibodies, in which variable domains of mAbs that target epitopes from multiple filoviruses were combined, for their capacity to neutralize viral infection across filovirus species. We found that bispecific combinations targeting EBOV and Sudan virus (another ebolavirus), provide potent cross-neutralization and protection in mice. Furthermore, trispecific combinations, targeting EBOV, Sudan virus, and Marburg virus, exhibited strong neutralization potential against all three viruses. These results provide important insights into multispecific antibody engineering against filoviruses and will inform future immunotherapeutic discoveries.

Filoviruses are negative-strand RNA viruses that cause severe hemorrhagic fever with case fatality rates of up to 90% in humans and nonhuman primates (1,2). There are five known ebolaviruses: Ebola virus (formerly "Ebola Zaire," EBOV 3 ), Res-ton virus, Taï Forest virus, Sudan virus (SUDV), and Bundibugyo virus. EBOV has been the most prevalent in terms of human infections, but both SUDV and Bundibugyo virus have caused large (Ͼ300 cases) outbreaks in the past (3).
With more than 28,000 suspected cases of infection, the 2014 -2016 EBOV disease epidemic in western Africa far exceeded the scale of any previous filovirus outbreak and underscored the need for filovirus pre-and post-exposure treatments. (4) To date, no therapeutic drug is approved, although several mAb mixtures are undergoing clinical trials. A number of studies have shown that mAb or mAb mixtures provide protective efficacy in nonhuman primates (NHPs) (5)(6)(7)(8). One mAb mixture, ZMapp TM (Mapp Biopharmaceutical), reversed the course of advanced Ebola virus disease in NHPs when provided 5 days after infection (5). A recent clinical study (PREVAIL II) indicated that although patients receiving ZMapp TM fared better than those receiving the previous standard of care, these differences within the small sampling of patients were below the threshold of statistical significance (9). Nonetheless, based on the strong trend of efficacy, the NIAID, National Institutes of Health and the Food and Drug Administration now consider ZMapp TM to be the standard of care. These results and more recent data showing that mAbs can protect NHPs from MARV challenge indicate that mAbs or mAb mixtures have strong therapeutic potential (10).
Until recently, there were few filovirus mAbs with demonstrated cross-neutralizing or cross-protective activity. This is likely due to the high degree of sequence variability across ebolaviruses in the envelope glycoprotein (GP), the primary target of neutralizing antibodies (11)(12)(13). Nonetheless, several mAbs from macaque immunizations or natural human infections have recently been shown to confer in vivo protection of rodents from multiple ebolaviruses (14 -19). The prefusion GP spike consists of three copies each of the surface subunit (GP1) and the transmembrane subunit (GP2) (20 -22). Regions of GP1, including its heavily glycosylated mucin-like domain (MLD), constitute the majority of the solvent-exposed surface. Cross-neutralizing GP1 mAbs typically target the glycan cap region (23,24). At the exposed region of the GP1-GP2 interface (the "base"), the GP2 fusion loop and surrounding residues provide other targets of cross-neutralizing mAbs (23). Given the sporadic nature of filovirus outbreaks and the inability to predict which viral species will be the causative agent, there is a strong potential benefit to cross-protective therapies.
We and others have been exploring multispecific antibody engineering strategies as a complementary approach to generation of cross-protective antibodies (25,26). We showed recently that combining two species-specific variable domains into a bispecific antibody (bsAb) confers cross-protection in mice for EBOV and SUDV (27). More recently, the specific combination of variable domains targeting broadly reactive (but non-neutralizing) epitopes on the viral surface with variable domains that disrupt critical interactions between the host receptor (Niemann Pick C1) and the endosomally exposed receptor-binding site (RBS) of GP1 resulted in pan-ebolavirusneutralizing activity (28 -30). Here we examine an extended panel of bsAbs targeting EBOV and SUDV and explore trispecific combinations (tsAbs) designed to provide activity against EBOV, SUDV, and MARV. The results illustrate that crossneutralization across diverse filoviruses is possible with appropriately engineered antibodies. Our results further reveal sites of vulnerability in the glycoprotein and add to the repertoire of cross-neutralizing antibodies.

Design, expression, and purification of multispecific antibodies
Previously we reported the construction and evaluation of bsAbs targeting the GP base epitope of EBOV and SUDV utilizing the scFv-Ig format (27). For these bsAbs, we employed the variable domains of two humanized variants of SUDV mAb 16F6 (F4 and E10) and the human EBOV mAb KZ52. We found that the most efficacious combination was fusion of the scFv from KZ52 to the C terminus of the F4 heavy chain (scKZ52-F4 HCC). This molecule provided potent cross-neutralization of both surrogate viral particles (vesicular stomatitis virus bearing the GP of EBOV or SUDV, VSV-GP(EBOV) or VSV-GP(SUDV), respectively) as well as authentic EBOV (Mayinga) and SUDV (Boniface). Furthermore, in two separate murine models of disease, scKZ52-F4 HCC afforded complete protection with post-exposure dosing.
We sought to explore alternative variable domains and bsAb formats to determine the effects on binding and inhibitory properties. Specifically, although the KZ52 mAb provided protection in mice and guinea pigs, and the variable domains, when integrated into the scFv-Ig fusions, provided protection in mice, in a single study, KZ52 was not protective in NHPs. To explore alternative EBOV-specific variable domains targeting the GP base epitope, we designed and generated scFv-IgG constructs utilizing the F4 IgG with scFv from c2G4, one of the neutralizing ZMapp TM components, as an alternative (Fig. 1A) (5). The c2G4 scFv was fused at the heavy chain C terminus (sc2G4-F4 HCC), light chain N terminus (sc2G4-F4 LCN), and light chain C terminus (sc2G4-F4 LCC). Previously we had determined that fusion of scFvs to the N terminus of the F4 IgG light chain abrogates activity, likely by sequestration of required interactions of the F4 heavy chain with SUDV GP.
To expand the potential scope of filovirus multispecific antibodies, we sought to integrate a third specificity toward MARV. The domain organization of MARV GP differs significantly from those of the ebolaviruses, including in sites of susceptibility for neutralization. The MARV GP mucin-like domain is proposed to sit lower on the trimer, likely occluding binding at the base region. However, the RBS is more exposed, and thus mAbs that bind the RBS have been shown to neutralize MARV GP (31)(32)(33). Such RBS-binding mAbs can cross-react with the corresponding regions on ebolavirus GP because the RBS is highly conserved across the family, but do not confer strong neutralization of ebolaviruses because the RBS is generally sequestered in the native form. Thus, targeting of the ebolavirus RBS requires intracellular delivery of RBS mAbs to the endosome, where viral membrane fusion occurs (28). Nonetheless, MARV mAb MR78 has been shown previously to inhibit MARV entry (32). Thus, we created a series of trispecific antibodies (tsAbs) in an scFv-scFv-IgG fusion containing the variable domains of MR78, KZ52, and F4 (Fig. 1B). Initial tsAb designs containing the F4 IgG as the central molecule with scFvs of KZ52 and MR78 appended as fusions to the N and/or C termini of the light and heavy chains were generated but did not exhibit neutralizing properties against VSV-GP(MARV) (see below). Thus, subsequent tsAb designs utilized an MR78 IgG core but with KZ52 and F4 scFvs added to the C termini of the light and heavy chains.

Engineered multispecific antibodies against filoviruses
Although the scFv-IgG format provides a straightforward design and expression strategy for generation of bsAbs, the long polypeptide linkers between variable domains on the scFv or between the scFv and IgG represent a potential liability for proteolytic degradation or immunogenic presentation. Furthermore, scFvs are known in some instances to oligomerize or be prone to aggregation because of inter-scFv association of the variable domains that can still dissociate from one another because they are connected by a long flexible linker. For EBOVspecific mAbs, our previous studies have explored mostly variable domains that target a single neutralizing epitope at the base of the GP, but other regions (such as the glycan cap) are also known sites of susceptibility (24). We therefore also generated bsAbs comprising the variable domains of F4 or E10 (SUDV-specific) fused to those of c13C6 (EBOV-specific, and a ZMapp TM component) in the dual variable domain (DVD-Ig) format ( Fig. 1C) (34). The DVD-Ig utilizes short human-derived peptide linkers to directly append one set of variable domains on top of another set within the IgG backbone. Presumably, the shorter linker length is less susceptible to proteolysis, and there is less inherent flexibility and, thus, aggregation because variable domain dissociation is less likely.
All bsAbs and tsAbs were expressed readily in human embryonic kidney 293 suspension cells and were purified by protein A chromatography. bsAbs and tsAbs migrated with the expected molecular weight by SDS-PAGE analysis and fragmented into the expected heavy and light chains upon reduction ( Fig. 1 and Fig. S2).

Binding studies and biochemical properties
Binding activities for bsAb and tsAbs were assessed initially by ELISA (Fig. 2) and then more quantitatively by biolayer interferometry (Fig. 3). For sc2G4-F4 bsAbs, the binding activity against both EBOV and SUDV GP was retained in all three of the tested formats (HCC, LCC, and LCN). ELISA with either EBOV or SUDV GP immobilized onto the well provided nanomolar EC 50 values. BLI studies, in which the bsAb was immobilized onto the sensor surface and then dipped into an analyte solution containing EBOV or SUDV GP, indicated that association with either GPs occurred in the 10 4 -10 5 /Ms range, and dissociation in the 10 2-10 3 /s range. The kinetic binding data could be described reasonably with a 1:1 model. However, because the bsAbs are bivalent in their ability to engage the antigen, and the GPs themselves are trimeric, it's likely that the binding stoichiometries are more complicated than a simple 1:1 model and thus the kinetic data represent an ensemble of interaction stoichiometries. Nonetheless, the k off /k on ratios ("apparent K D s") were in the range of 10 Ϫ7 -10 Ϫ8 M. These values were consistent with the approximate affinities that we have determined previously for F4 (2.5 ϫ 10 Ϫ7 M) and c2G4 (2.2 ϫ 10 Ϫ7 M), indicating that the affinity of the variable domains within the bsAb was not affected (35).
A tsAb containing F4 as the core IgG with scFvs of KZ52 and MR78 as fusions to the heavy chain C terminus and light chain N terminus, respectively, ("scKZ52(LCN)-scKZ52(HCC)-F4") retained binding activity toward EBOV, SUDV, and MARV ( Figs. 1 and 4). However, this tsAb and other tsAbs bearing F4 as the central IgG and scFvs for MR78 and KZ52 failed to neutralize VSV-GP(MARV) at 270 nm (Fig. 4). These results suggest that conversion of the MR78 variable domains into the scFv format results in loss of proper assembly of the variable domains to faithfully recapitulate the MR78 activity, despite the ability to bind MARV GP.

Engineered multispecific antibodies against filoviruses
In contrast, a tsAb bearing MR78 as the core IgG with scFvs of F4 and KZ52 appended as C-terminal fusions to the heavy and light chains, respectively, ("scF4(HCC)-scKZ52(LCC)-MR78") was able to bind and neutralize all three GPs. BLI measurements provided apparent K D measurements in the doubledigit nanomolar range against MARV and EBOV and ϳ650 nM
To examine whether all three specificities on scF4(HCC)-scKZ52(LCC)-MR78 could be engaged simultaneously, a threestep sequential binding experiment was performed in which immobilized tsAb was sequentially dipped in solutions containing GP(EBOV), GP(MARV), and GP(SUDV) (Fig. 5D). All three antigens could be bound simultaneously.
The c13C6ϳE10 DVD-Ig bound both GP(EBOV) and GP(SUDV), but, surprisingly, the c13C6ϳF4 DVD-Ig did not bind to the GP(SUDV) (Figs. 2 and 3). The basis for this difference in activity is not clear, and is unanticipated because F4 and E10 are highly related to one another in terms of sequence. Nonetheless, kinetic binding data indicate that affinity for both components was again retained for the c13C6ϳE10 DVD, with 5.3 ϫ 10 Ϫ8 M for GP(SUDV) and 3.8 ϫ 10 Ϫ9 M for GP(EBOV) (previously reported to be 2.1 nM by Doranz and co-workers (36).

Neutralization profiles
We next sought to explore the capacity of bsAbs and tsAbs to inhibit cell entry mediated by multiple filovirus glycoproteins. For this purpose, we utilized recombinant vesicular stomatitis virus particles containing filovirus GP in place of the native glycoprotein G (rVSV-GPs). All rVSV-GPs contained an enhanced GFP (eGFP) in the genome to facilitate quantification of infection events by fluorescence. The rVSV-GPs have been shown previously to provide faithful recapitulation of GP-mediated cell attachment and viral fusion (37). Furthermore, neutralization of rVSV-GPs has been shown previously to be a highly predictive measure of antibody neutralization of authentic pathogens but can be manipulated under routine (BSL2) laboratory conditions (18,27,28).
All three variants of the scFv-IgG fusions of 2G4 and F4 exhibited strong neutralization potential against rVSV-GP-(SUDV) and rVSV-GP(EBOV), similar to a mixture consisting of a stoichiometric 1:1 mixture of the monospecific parental antibodies (Fig. 6, A and B). The sc2G4-F4 HCC construct had the strongest neutralization potential against both rVSV-GPs, whereas activity of sc2G4-F4 LCC was lower for rVSV-GP(EBOV) despite comparable binding affinity relative to the two other sc2G4-F4 scFv-IgG variants. A possible explanation for this discrepancy is that the location of the scFv on the light chain C terminus somewhat occludes the potential for intraspike cross-linking by the c2G4 scFv. Notably, we and others have found that Fab fragments of base-targeting filovirus mAbs have a much lower neutralization potential than the corresponding IgGs, suggesting that the bivalent cross-linking contributes to the neutralization activity (35). sc2G4-F4 HCC had comparable IC 50 values against both rVSV-GPs relative to individual parental antibodies as well as to the respective 1:1 mixture. However, the activity of sc2G4-F4 LCN and sc2G4-F4 LCC was somewhat diminished against rVSV-GP(EBOV) relative to the c2G4 parental control. This diminution in activity was not observed for rVSV-GP(SUDV) for any of the bsAbs compared with the F4 monospecific control. The two monospecific controls were orthogonal in their activities, with F4 having no activity against rVSV-GP(EBOV) and c2G4 likewise being inactive against rVSV-GP(SUDV). The c13C6ϳE10 DVD-Ig was potently neutralizing against rVSV-GP(SUDV), albeit with a reduction in activity relative to the WT parental E10 IgG as well as the respective comparator mixture (Fig. 6D). Neither c13C6 nor the DVD-Ig showed any significant neutralization activity against rVSV-GP(EBOV) (Fig. 6C). However,

Engineered multispecific antibodies against filoviruses
this result was expected and consistent with previous studies of c13C6. c13C6 engages the glycan cap epitope and is non-neutralizing but indispensable for ZMapp TM in vivo protection. The specific mechanism of c13C6-mediated protection is unclear but may involve Fc-mediate function for infected cell or virus clearing.
The scF4(HCC)-scKZ52(LCC)-MR78 tsAb neutralized all three rVSV-GPs, albeit with somewhat reduced potency against rVSV-GP(EBOV) relative to the parental KZ52 mAb (Fig. 7, A-D). IC 50 values against all three viruses were in the nanomolar range, similar to those observed for the mixture consisting of the monospecific parental antibodies, despite the fact that SUDV binding had ϳ10-fold lower affinity. When the tsAb was tested in a microneutralization assay with authentic EBOV, SUDV, and MARV, high levels of neutralization against all three pathogens were observed at concentrations of 500 nM, with diminishing levels of neutralization at lower tsAb concentrations (Fig. 7, E and F). However, the parental F4 and KZ52 mAbs were strongly neutralizing (Ͼ70%) against their respective authentic pathogens at concentrations as low as 2 nM. Levels of neutralization against authentic MARV were similar among the parental MR78 and the tsAb.

In vivo activity of E10ϳ13C6 DVD Ig
The protective capacity of the E10ϳ13C6 DVD was tested in two separate post-exposure mouse models. For EBOV, WT C57BL/6 mice were challenged with a lethal dose of mouseadapted EBOV (maEBOV) and then treated 1 day post-infec-tion with 100 g of bsAb. For the SUDV challenge, we utilized a previously reported model in which type I ␣/␤ interferon receptor knockout (IFNAR Ϫ/Ϫ ) mice were challenged with human lethal Sudan virus (Boniface). bsAb treatment (500 g/dose) was provided on days 1 and 5 post-exposure. In each case, the monospecific controls E10 and 13C6 were included, as was a 1:1 mixture of these two mAbs for comparison. For the SUDV challenge, because 13C6 exhibits some SUDV activity, the monospecific EBOV mAb 6D8 was included as a further control. As shown in Fig. 8, A and B, E10ϳ13C6 DVD-Ig provided 80% protection against maEBOV and 70% protection against SUDV; however, protection against SUDV was not statistically significant relative to the control mAb 6D8 (30%), with a group size of n ϭ 10. In both models, weight loss in E10ϳ13C6 DVD-Ig-treated mice was observed, a clinical sign of illness. However, in both cases, the aggregate weight loss was followed by weight gain after day ϳ10, indicating recovery from disease. For SUDV, this weight loss trend was again indistinguishable from the negative control 6D8. For both viruses, the mixture of E10 and 13C6 provided significant protective efficacy relative to controls (100% and 90% protection in maEBOV and SUDV, respectively), but mice receiving the combination therapy experienced no (maEBOV) or little weight loss (SUDV). The monospecific SUDV control E10 afforded no activity against maEBOV, with all mice losing weight until succumbing to the disease. However, E10 was 90% protective against SUDV, with only moderate weight loss, as reported previously. 13C6 was

Engineered multispecific antibodies against filoviruses
100% protective against maEBOV (as reported previously), and 60% protective against SUDV, but again indistinguishable from the negative control mAb 6D8 in terms of survival and weight loss. Together, these results suggest that E10ϳ13C6 DVD Ig is strongly protective against EBOV but does not confer a statistically significant survival advantage for SUDV relative to the negative control mAb 6D8.

Discussion
Here we have explored newly designed bsAb and tsAbs for neutralization of multiple filoviruses. These reagents are complementary to the existing set of cross-neutralizing mAbs from NHP immunization and human infection (14 -19, 27, 28). Furthermore, the structure-activity relationship studies reported here with different bsAb and tsAb formats provide novel insights into requirements for cross-neutralization and crossprotection from filoviruses that can be utilized for future mAb engineering efforts.
We found here and previously that many bsAb combinations employing combinations of SUDV mAb F4 and base-binding EBOV mAbs (KZ52 and 2G4) afford potent cross-neutralization of these two filoviruses (27). These studies further confirm the base structural subdomain as an important site of vulnerability for at least SUDV and EBOV. Recently, human mAbs that bind slightly higher on the prefusion spike (e.g. ADI-15878, ADI-15742, and ADI-15946) have been shown to afford broad or even panebolavirus-neutralizing activity (18). Mechanistically, binding in this region likely sequesters critical transformations of GP during viral entry. For example, ADI-15946 stabilizes EBOV GP to proteolysis by CatL, a critical process that exposes the RBS for binding to NPC1. Furthermore, KZ52 may sequester critical membrane fusion events within endosomes. The work described here, along with structural data on ADI-15878, ADI-15742, and ADI-15946, suggest that binding of GP in this region is enough to confer neutralization and protection potential, at least in rodents.

Engineered multispecific antibodies against filoviruses
We further explored the potential for a bsAb combination employing an SUDV GP base binder (E10) linked to an EBOV GP glycan cap binder (13C6) in the DVD-Ig format. Although this bis-mAb reflected aspects of both parental mAbs in binding and neutralization profiles, it provided in vivo efficacy only against EBOV. Therefore, the precise combination of mAbs and the bispecific format into which they are integrated can have nonobvious effects for in vivo efficacy. Some degree of trial and error, as with all bsAb design strategies, is required to identify optimal combinations.
Results with tsAbs indicate that, in principle, neutralization against disparate members of filoviruses is possible with a single agent. However, it seems likely that the parameters required for neutralization of MARV differ significantly from any of the ebolaviruses. Only a single non-neutralizing mAb, FVM02p, has been described to cross-react across multiple ebolaviruses and MARV (17). Overall, MARV GP is only ϳ30% identical to EBOV (whereas the GP of SUDV, the most distant ebolavirus from EBOV, is ϳ55% identical). Furthermore, there are distinct differences in the domain organization and spatial location of the MLD between the prefusion GP of EBOV and MARV. It has been reported that the MLD sits lower on the viral spike in MARV, likely sequestering base epitopes but leading to a more exposed RBS (33). Thus, true pan-filovirus activity may require combination of multiple mAbs, some specific to Ebola viruses

Engineered multispecific antibodies against filoviruses
but others specific for Marburg viruses, either as a mixture or as engineered multispecific Abs. Our results further indicate that engineering of tsAbs with three distinct functional specificities is, in fact, possible with the scFv-scFv-IgG format. However, we found that only particular combinations yielded productive tsAbs.
The bsAbs and tsAbs reported here may have immunotherapeutic potential, especially when included in mixtures with other cross-neutralizing mAbs. Overall, for viral immunotherapy, it seems advantageous to target multiple epitopes to mitigate viral escape by a single point mutation and to leverage potential synergism in mechanisms of action. In this context, it seems that a potentially tractable approach to multiepitope targeting with broad mixtures would be to combine multiple appropriately engineered bsAbs or tsAbs. Furthermore, for cases where bsAbs provide equivalent activity to a mixture consisting of the individual mAbs, inclusion of those mAbs as part of bsAb allows the use of both specificities but without the manufacturing burden of having to produce both molecules. In theory, such a manufacturing burden could be tractable for combination of two mAbs, but combination of four or more mAbs may be too onerous to be feasible on a large scale. Therefore, multispecific antibody engineering approaches described here may be implemented to reduce the manufacturing complexity of producing a mixture.
Multispecific mAbs have been utilized extensively in oncology, but their applications in viral immunotherapy are only now emerging (25). At present, the true immunotherapeutic potential of multispecific, engineered mAbs in relation to combinations of traditional mAbs remains to be determined. Multispecific mAbs contain engineered, non-natural segments that could potentially affect critical parameters such as stability and in vivo half-life and could elicit an anti-therapeutic antibody response by the host. However, the advantages of multispecific mAbs include the ability to target multiple epitopes within a single agent and the capacity to engender properties within an engineered bi-or trifunctional molecule that is not possible with either component alone. A good example of this latter principle is the development of "Trojan horse" bsAbs that engage the ebolavirus GP RBS deep within the endosomal pathway (18). The results with bsAbs and tsAbs presented here, and in other reports for filoviruses and other viral pathogens (25,26), indicate that multispecific antibodies may exhibit enhanced properties relative to canonical monospecific mAbs that are particularly beneficial for immunotherapy.

Antibody expression and purification
To facilitate the expression of multispecific antibody molecules, the respective single-chain variable fragment (scFv) as well as DVD fusions were subcloned into the respective pMAZ-IgH and pMAZ-IgL vectors developed by Mazor et al. (38) (Fig.  S1). Subsequently, the respective pMAZ-IgH and pMAZ-IgL vectors encoding each antibody were co-transfected into Freestyle TM -293F suspension-adapted human embryonic kidney 293 cells using linear polyethyleneimine (Polysciences, Warrington, PA). Cell cultures were incubated at 37°C and 8% CO 2 for 6 days post-transfection. Cleared cell supernatants were then applied to a protein A affinity column (ϳ1 ml of packed beads per 600 ml of culture) (Thermo Scientific). Antibodies were purified using the Gentle Antibody Elution System (Thermo Scientific) according to the manufacturer's instructions and subsequently exchanged into 150 mM HEPES (pH 7.4) and 200 nM NaCl.

BLI
The OctetRed TM system (ForteBio, Pall LLC) was used to determine the binding properties of IgGs and DVD-Igs. Antihuman Fc capture sensors were used for initial Ab loading. For single-phase binding experiments, global data fitting to a 1:1 binding model was used to estimate values for the k on (association rate constant), k off (dissociation rate constant), and K D (equilibrium dissociation constant). For double-phase binding experiment, the Ab was first immobilized on an Fc sensor and then allowed to equilibrate in a solution containing the first antigen. The sensor was then transferred to a second solution containing the second antigen.

Virus neutralization assays
Recombinant vesicular stomatitis Indiana viruses (rVSVs) expressing eGFP and EBOV GP in place of VSV G have been described previously (28,37). Similar rVSVs expressing eGFP and representative GP proteins from SUDV and MARV were generated as above. The infectivities of rVSVs were measured by automated enumeration of eGFPϩ cells (infectious units) using a CellInsight CX5 imager (Thermo Fisher) at 12-14 h post-infection. For Ab neutralization experiments, pretitrated amounts of rVSV-GP particles (multiplicity of infection Ϸ 1 infectious unit/cell) were incubated with increasing concentrations of test Ab at room temp for 1 h prior to addition to cell monolayers in 96-well plates. Viral infectivities were measured as above.

Mouse challenge experiments
Research was conducted under an IACUC-approved protocol in compliance with the Animal Welfare Act, Public Health Service Policy, and other federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011.
Female C57BL/6 mice (6 -14 weeks old) (The Jackson Laboratory) were challenged via the intraperitoneal route (I.P.) with 1000 pfu of maEBOV. Mice were treated 1 day post-challenge I.P. with 100 g of antibody or PBS.
Male and female type 1 IFN ␣/␤ receptor knockout mice (type 1 IFN␣/␤ R Ϫ / Ϫ ) purchased from The Jackson Laboratory (4 -14 weeks of age) were utilized in these experiments. Mice were challenged I.P. with a target dose of 1000 pfu of WT SUDV and treated I.P. with 500 g of the indicated mAb at day ϩ1 and day ϩ5.
Following all challenges, mice were observed daily for clinical signs of disease and lethality. Daily observations were increased Engineered multispecific antibodies against filoviruses to a minimum of twice daily while mice were exhibiting signs of disease. Moribund mice were humanely euthanized on the basis of IACUC-approved criteria.


extfieldsofstudy: Physics Medicine

s2fieldsofstudy: Biology Engineering

text:
Jet collisions and vortex reversal in the human left ventricle

Unnatural dynamics of the notorious vortex in the left ventricle is often associated with cardiac disease. Understanding how different cardiac diseases alter the flow physics in the left ventricle may therefore provide a powerful tool for disease detection. In this work, the fluid dynamics in the left ventricle subject to different severities of aortic regurgitation is experimentally investigated by performing time-resolved particle image velocimetry in a left heart duplicator. Diastolic vortex reversal was observed in the left ventricle accompanied by an increase in viscous energy dissipation. Vortex dynamics and energy dissipation may provide useful insights on sub-optimal flow patterns in the left ventricle.

I. Introduction
The left ventricle (LV) is the heart's powerhouse, responsible for supplying each and every tissue throughout the body with oxygenated blood. During its filling phase (diastole), a vortical structure is known to develop in the LV, which has inspired a number of fluid dynamic studies over the past two decades owing to its potential for early detection of disease. This vortex facilitates the ejection of blood (systole) by redirecting the eccentric inflow toward the LV outflow tract (Kilner et al., 2000) with some evidence that it also minimizes energy dissipation (Pedrizzetti & Domenichini, 2005) and blood residence time (Hendabadi et al., 2013). Unnatural dynamics of this vortex may be correlated with disease (Narula, Vannan, & DeMaria, 2007), thus inspiring methods to characterize the behavior of a "healthy" vortex such as verifying the energy dissipation characteristics of the flow (Pedrizzetti & Domenichini, 2005;Stugaard et al., 2015;Raymondet, Kadem, & Di Labbio, 2016; and defining a left ventricular vortex formation time (Gharib et al., 2006). This work focuses on the dynamics of this notorious vortex in the presence of a particularly prevalent valvular disease, namely aortic regurgitation.
Aortic regurgitation (AR) is a condition where the aortic valve does not adequately close. In such a case, LV filling partly occurs from leakage through the aortic valve, leading to a disruption of the formation of the diastolic vortex due to the interaction between the mitral inflow and the emergent aortic regurgitant jet (Fig. 1). AR itself is quite common, with mild AR or worse having an estimated prevalence of 5.2% in the adult population over age 40 in the USA (Singh et al., 1999). Of particular interest here is the case of chronic AR, where the left ventricle gradually responds to the persistent regurgitation by dilating in an attempt to accommodate the regurgitant volume and maintain a healthy forward stroke volume, while the peak aortic pressure may remain relatively unchanged or increase (Bekeredjian & Grayburn, 2005;Stout & Verrier, 2009). Figure 1: Schematic of the interaction between a regurgitant jet emanating from the aortic valve (AV) and the diastolic vortex developing from ordinary mitral valve (MV) inflow as the regurgitation worsens based on the results of this study (see Sec. III for more detail).

II. Methods
The intraventricular flow in the context of chronic AR was studied by experiment in a double-activation left heart duplicator (Fig. 2). The duplicator consists of elastic silicone (SILASTIC RTV-4234-T4, The Dow Chemical Company; USA) models of the left atrium, left ventricle and aorta, having refractive indices of 1.41 ± 0.01. The ventricle (model made available in Appendix A) is encased in a hydraulic chamber with its activation being controlled by a piston-cylinder-type assembly. The left atrium is directly connected to an elevated reservoir, allowing atrial filling to occur passively. LV filling occurs in two distinct phases: ventricular relaxation (E wave) and atrial contraction (A wave). At the start of filling, the hydraulic piston pulls back, expanding the ventricle as it fills through the mitral valve (23 mm Perimount Magna Ease, Edwards Lifesciences; USA). As the piston attains its maximal backward stroke, a servomotor compresses the atrium to provide the A wave. This begins the piston's forward stroke, hydraulically compressing the ventricle to enable ejection through the aortic valve (25 mm Perimount Theon RSR, Edwards Lifesciences; USA) up to the reservoir, completing the cardiac cycle. The reader is referred to the supplementary information contained in Appendix A.1 for further technical information regarding the system. The working fluid was a mixture of 60% water and 40% glycerol by volume, having a refractive index of 1.39 and giving a measured dynamic viscosity (4.2 cP) and density (1100 kg/m 3 ) relatively similar to those of blood.
The term "double-activation" refers to independent activation of the LV and left atrium to respectively produce the E and A waves of filling. This is critical in experimentally simulating AR, as most left heart duplicators produce the A wave by providing an additional ventricular expansion using the hydraulic piston, which would induce prolonged, non-realistic, regurgitation.
AR was introduced by pulling apart graduated rods hooked under each aortic valve leaflet to restrict valve closure to a centralized orifice while allowing the leaflets to open freely (refer to Fig. 3 for a schematic and to the supplementary information in Appendix A.1 for more details). Five different severities and one "healthy" scenario were investigated by changing the regurgitant orifice area (ROA) to be 0 (normal), 0.10 (mild), 0.18 (moderate-1), 0.25 (moderate-2), 0.52 (severe-1) and 0.78 cm 2 (severe-2), in accordance with clinical guidelines (Nishimura et al., 2014). These orifices respectively represent 0, 3.3, 5.9, 8.5, 17.2 and 26.1% of the fully open geometric aortic valve area (3.00 cm 2 ). The physical requirement of maintaining a constant forward stroke is equivalent to increasing the total stroke such that the volume contributed from mitral inflow remains constant. As such, the mitral inflow volume was held constant at 64 ± 4 mL, with the A wave corresponding to 21 ± 4 mL, at a heart rate of 70 bpm (T = 0.857 s), while the peak aortic pressure was maintained at 121 ± 5 mmHg. A normalized time, t * = t/T , is here defined with t * = 0 marking the start of the ejection phase, t * = 0.44 the beginning of the filling phase (end of ejection), and t * = 1 the end of the cycle. The flow circuit was seeded with polyamide particles (mean diameter: 50 µm, density: 1030 kg/m 3 ), to capture the velocity field by 2D time-resolved particle image velocimetry. The particles were illuminated using a doublepulsed Nd:YLF laser (LDY301, Litron Lasers; England) forming a 1 mm thick laser sheet at the test section. The LV flow domain was captured at a recording rate of 400 Hz (double-frame images 700 µs apart) at the full resolution of 1632 × 1200 pixels using a CMOS camera (Phantom v9.1, Vision Research Inc.; USA). The flow was recorded over one cycle in the plane crossing both the mitral and aortic valves and the ventricle apex, a view often used in clinical practice to evaluate AR (Lancellotti et al., 2013). The velocity fields were computed in DaVis 8.2 (LaVision GmbH; Germany) using a multi-pass cross-correlation algorithm with decreasing window size (64 × 64 pixels down to 16 × 16 pixels, using 50% overlap). The final spatial resolution was 0.52 × 0.52 mm. The total uncertainty in the velocity field was estimated to be below 5% with respect to the maximum pointwise velocities observed in the healthy (1.70 m/s) and severe (1.66 m/s) cases, based on the major uncertainties described in Raffel et al. (2007) and Adrian and Westerweel (2011). Figure 3: Schematic of the mechanism used to impose aortic regurgitation in the cardiac duplicator. Graduated rods hooked under each aortic valve leaflet were pulled apart to maintain a centralized regurgitant orifice area (ROA) for five cases. The rods were removed to simulate the healthy flow scenario.

A. Replication of healthy left ventricular flow
The experiment replicates the healthy intraventricular flow rather well. A vortex ring rolls up from the shear layer of the mitral inflow, one side of which dissipates against the ventricular wall as the other side imparts a swirling motion to the entire ventricular volume (Pedrizzetti & Domenichini, 2005); see Fig. 4 and Video 1 in Appendix A.2. Virtual particles released at the start of diastole exhibit a clockwise swirl and are readily aligned for ejection by the start of systole (Fig. 4, right panel). The dimensionless vortex formation time (Gharib et al., 2006) is calculated to be 4.1, falling in the "healthy" range.

B. Vortex reversal and flow behavior
As the AR severity worsens, the regurgitant jet readily interacts with the mitral inflow. The two jets remain rather confined to the ventricle walls and each generate their respective vortices as their shear layers roll up. The two vortices effectively compete for space in the LV, with the regurgitant vortex gradually gaining the upper hand and occupying more of the ventricular volume as the severity worsens. Initially, in the mild and moderate-1 cases (ROA = 3.3 and 5.9%), the regurgitant jet emanates after the mitral inflow. In the former, the healthy vortical flow does not seem to be significantly disturbed as the small regurgitant volume is simply entrained by the mitral vortex generated during the E wave, limiting its progression into the ventricle. However, filling from the mitral valve is noticeably less forceful, having diminished velocity magnitudes, and the vortex core is ultimately unable to set up in the ventricle's center. In the latter, when the regurgitant jet emanates, it pushes the mitral vortex toward the wall, allowing the regurgitant jet to penetrate deeper into the ventricle and establish a counter-rotating vortex resembling the middle schematic of Fig. 1. In the moderate-2 case (ROA = 8.5%), the regurgitant jet and the mitral jet begin nearly at the same time; Video 2 in Appendix A.3. With the mitral inflow volume exceeding the regurgitant volume, the mitral jet sets up the more dominant vortex. The subsequent flow in this scenario is rather interesting as it represents a critical condition dependent on the timing of the two emerging jets, resembling either the moderate-1 case (Fig. 1, middle schematic) or the severe cases (Fig. 1, rightmost schematic). In the severe cases (ROA = 17.2 and 26.1%), the regurgitant jet precedes the mitral inflow and establishes a counter-rotating vortex occupying the base of the ventricle; Video 3 in Appendix A.4. This is demonstrated in Fig. 5 (left panel), where the regurgitant vortex imparts a completely reversed swirling motion to the flow at the base of the ventricle by mid-diastole and restricts further progression of the mitral inflow. In this case, particles released at the start of diastole (t * = 0.44) are not favorably oriented for ejection, some of which follow a reversed swirling path compared to the healthy scenario (Fig. 5, right panel). To illustrate this vortex reversal further, in Fig. 6, the temporal evolution of the circulation in the ventricle per unit area is plotted, showing a general progression toward positive values (counter-clockwise rotation) with regurgitation severity.

C. Energy loss characteristics
Following recent interest (Pedrizzetti & Domenichini, 2005;Stugaard et al., 2015), the rate at which energy is dissipated by viscous stresses in the flow is illustrated in Fig. 7. This quantity could be seen as a measure of the efficiency of the intraventricular flow to conserve inflowing kinetic energy, suggesting a greater work input requirement from the heart to compensate for greater losses in the flow. In 2D, the total viscous energy dissipation rate (VED) is given below (units of power per unit depth) with µ being the dynamic viscosity. Characteristically, this quantity is zero in the case of a vortex described by solid body rotation and becomes appreciable in regions of elevated longitudinal or transverse strain rate (as in jets and shear layers).
A clear overall increase is observed with regurgitation severity. This is consistent with the in vivo results reported by Stugaard et al. (2015), despite the limitations associated with their use of vector flow mapping to acquire velocity fields (Pedrizzetti & Sengupta, 2015). The initial climb of the dissipation rate is associated with the high strain rates accompanying the mitral and regurgitant jets, as well as with mild turbulent fluctuations accompanying the regurgitant jet. The peak corresponds more or less to the end of the E wave. The following decay is associated with the establishment of a vortical motion in the ventricle, whose core somewhat approximates rigid body rotation. Repeating the experiment 10 times for each case and comparing successive curves by p-value calculations using two- Figure 6: The circulation per unit area (or spatial mean vorticity) progressively tends toward positive values with regurgitation severity, associated with a general counter-clockwise sense of rotation.
way ANOVA, distinction between the values of total VED is confirmed at a 95% confidence level (p max = 2.4 · 10 −7 between the healthy and mild cases). Integrating under these curves for one cycle, the energy loss per unit depth can be seen to almost linearly increase with ROA (Fig. 8). It should be noted that although the peak VED in the healthy scenario is higher than that of the mild case, the energy loss is smaller owing to the prolonged effect of the perturbation imparted by the regurgitation. It appears that yet again the vortical motion in the healthy LV represents an optimal flow condition in the sense of energy conservation.

IV. Conclusion
These results suggest primarily that progressive vortex reversal in the LV may naturally arise with chronic AR. The potential of the total viscous energy loss per cycle to evaluate AR severity is also highlighted, remarkably showing an almost linear relationship between energy loss and ROA (Fig. 8), despite the overall nonlinear nature of the flow. This further suggests the energy loss to be an excellent indicator in the case of AR, being directly related to the ROA itself which is difficult to evaluate in practice.
Evidently, there are many factors that may alter the intraventricular flow that have not been investigated here. These include ventricular geometry for instance, as well as the nature of the regurgitant orifice and jet, with angle and eccentricity being perhaps the most influential. The fluid dynamic phenomena demonstrated in this work are based on an idealized model left ventricle and it is not yet clear whether they may be generalized to all left ventricular geometries. Furthermore, while the two-dimensional plane selected for study is the plane most commonly used in clinical practice to assess AR via color Doppler, it may not be the case that the viscous energy loss exhibits such a linear monotonic increase when considering the full three-dimensional flow. These authors believe that further research is needed to consider both geometrical differences and three-dimensionality, which this group is actively working to achieve. Additionally, we would like to stress that in the case of AR, in vivo velocity field data for a comprehensive set of severities, for which there is considerable need, will allow for better scrutiny of the aptitude of vortex and energy characteristics to assess AR severity against the current metrics. of his laboratory facilities (particularly DaVis 8.2 for this article). Additionally, the authors would like to thank the participants of the 8th International Bio-Fluid Symposium (Pasadena, CA; February, 2016) and the 69th Annual Meeting of the APS Division of Fluid Dynamics (Portland, OR;November, 2016) for their constructive comments and fruitful insight.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Medicine

text:
Potential Anti-Inflammatory and Anti-Cancer Properties of Farnesol

Farnesol, an acyclic sesquiterpene alcohol, is predominantly found in essential oils of various plants in nature. It has been reported to exhibit anti-cancer and anti-inflammatory effects, and also alleviate allergic asthma, gliosis, and edema. In numerous tumor cell lines, farnesol can modulate various tumorigenic proteins and/or modulates diverse signal transduction cascades. It can also induce apoptosis and downregulate cell proliferation, angiogenesis, and cell survival. To exert its anti-inflammatory/anti-oncogenic effects, farnesol can modulate Ras protein and nuclear factor kappa-light-chain-enhancer of activated B cells activation to downregulate the expression of various inflammatory mediators such as cyclooxygenase-2, inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin-6. In this review, we describe the potential mechanisms of action underlying the therapeutic effects of farnesol against cancers and inflammatory disorders. Furthermore, these findings support the clinical development of farnesol as a potential pharmacological agent in clinical studies.

Plants
Scientific Name Usages Ref.

Citronella
Cymbopogon nardus Source of soap, candles and incense, perfume, cosmetics, and flavoring [5] Lemon Grass Cymbopogon citratus Sterilization Relieves sweating, fever, abdominal pain, and controls skin secretions [5] Tuberose Polianthes tuberosa L. Source of perfume [5,6] Cyclamen Cyclamen persicum [7] Rose Rosa hybrida Source of perfume [8] Neroli Citrus aurantium subsp. amara Source of perfume [9] Balsam Source of medicinal products, fragrances for varnishes and lacquers, air freshener perfume, and natural remedy for skin rashes [5] Musk Abelmoschus moschatus Source of perfume [6]   One of the processes that predominantly drive tumor progression is inflammation. Although acute inflammation is part of the earliest responses to tissue damage, and it helps to counter against the invasion of foreign pathogens, chronic inflammation can create an environment that supports tumor initiation and development [40,41]. Chronic inflammation in the tumor microenvironment also allows for tumor invasion and metastasis to occur as pro-inflammatory mediators can provide signal to tumor cells to extravasate into the stroma [41]. Additionally, chronic inflammation can also drive major diseases such as atherosclerosis, asthma, and Alzheimer's disease [41]. The mechanism(s) by which chronic inflammation promotes these diseases, especially cancer, is through the continuous production of cytokines and chemokines, such as tumor necrosis factor alpha (TNFα), interleukin (IL)-1, IL-6, and C-X-C chemokine receptor type 4, through the constitutive activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is a pro-inflammatory transcription factor [40,41]. The inhibition of the pro-inflammatory NF-κB signaling pathway in various cancers has proven to effectively inhibit tumor growth, invasion, and metastasis; for example in hepatocellular carcinoma, colorectal, breast, and prostate cancer [42]. However, a total inhibition of inflammation can also worsen cancer prognosis, as noted from the use of TNFα blockers resulting in an increased risk of lymphoma development [40]. Therefore, regulation of inflammation in anti-cancer treatment should strive to ameliorate chronic inflammation to maintain the beneficial acute inflammatory state. Since farnesol has been shown to be able to regulate inflammation in several diseases and also inhibit tumor growth in various cancers, the inflammation-modulatory and anti-cancer effects of farnesol that have been reported in prior studies have been briefly summarized in this review.

In Vitro Pro-Inflammatory Effects of Farnesol
Farnesol has been found to stimulate the immune system response through upregulating the expression of pro-inflammatory genes such as IL-6, TNF-α, and cyclooxygenase-2 (COX-2) in human lung adenocarcinoma cells and murine macrophage cells ( Figure 3) [15,16]. These genes are One of the processes that predominantly drive tumor progression is inflammation. Although acute inflammation is part of the earliest responses to tissue damage, and it helps to counter against the invasion of foreign pathogens, chronic inflammation can create an environment that supports tumor initiation and development [40,41]. Chronic inflammation in the tumor microenvironment also allows for tumor invasion and metastasis to occur as pro-inflammatory mediators can provide signal to tumor cells to extravasate into the stroma [41]. Additionally, chronic inflammation can also drive major diseases such as atherosclerosis, asthma, and Alzheimer's disease [41]. The mechanism(s) by which chronic inflammation promotes these diseases, especially cancer, is through the continuous production of cytokines and chemokines, such as tumor necrosis factor alpha (TNFα), interleukin (IL)-1, IL-6, and C-X-C chemokine receptor type 4, through the constitutive activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is a pro-inflammatory transcription factor [40,41]. The inhibition of the pro-inflammatory NF-κB signaling pathway in various cancers has proven to effectively inhibit tumor growth, invasion, and metastasis; for example in hepatocellular carcinoma, colorectal, breast, and prostate cancer [42]. However, a total inhibition of inflammation can also worsen cancer prognosis, as noted from the use of TNFα blockers resulting in an increased risk of lymphoma development [40]. Therefore, regulation of inflammation in anti-cancer treatment should strive to ameliorate chronic inflammation to maintain the beneficial acute inflammatory state. Since farnesol has been shown to be able to regulate inflammation in several diseases and also inhibit tumor growth in various cancers, the inflammation-modulatory and anti-cancer effects of farnesol that have been reported in prior studies have been briefly summarized in this review.

In Vitro Pro-Inflammatory Effects of Farnesol
Farnesol has been found to stimulate the immune system response through upregulating the expression of pro-inflammatory genes such as IL-6, TNF-α, and cyclooxygenase-2 (COX-2) in human lung adenocarcinoma cells and murine macrophage cells ( Figure 3) [15,16]. These genes are controlled by NF-κB, which can also regulate the expression of a variety of genes involved in the immune-inflammatory response, including those related to pro-inflammatory activity [43]. NF-κB expression is regulated by nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκB-α) [43]. Farnesol can reduce the level of IκB-α protein, leading to an increase of the immune response in human lung adenocarcinoma cells (H460) ( Table 2) [16]. In addition, farnesol can induce the activation of mitogen-activated protein kinase (MAPK) signaling pathways, especially activation of MEK1/2-ERK1/2 [16]. Generally, activation of the MEK-ERK signaling pathway is a pro-survival signal, but activation of the MEK1/2-ERK1/2 pathway can also result in the induction of apoptosis [16]. Farnesol can induce phosphorylation of p65, which is dependent on activation of the MEK1/2-ERK1/2 signaling pathway in human lung adenocarcinoma cells (H460) [16], leading to increased p65 activity and chemokine ligand 3, IL-1, and COX-2 expression [16]. In addition, farnesol has shown to exert its pro-inflammatory effect by increasing the levels of IL-6, TNF-α, and COX-2 in macrophage cells (RAW 264.7) ( Table 2) [15]; these effects were markedly augmented when farnesol was combined with zymosan [15]. In an in vitro study carried out with primary murine splenocytes, farnesol treatment did not have an anti-inflammatory effect on the cells as it did not cause a significant change in the ratio of Th2/Th1 cytokines, as well as IL-10 and IL-2 production respectively, as compared to other sesquiterpenoid compounds [44]. controlled by NF-κB, which can also regulate the expression of a variety of genes involved in the immune-inflammatory response, including those related to pro-inflammatory activity [43]. NF-κB expression is regulated by nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκB-α) [43]. Farnesol can reduce the level of IκB-α protein, leading to an increase of the immune response in human lung adenocarcinoma cells (H460) ( Table 2) [16]. In addition, farnesol can induce the activation of mitogen-activated protein kinase (MAPK) signaling pathways, especially activation of MEK1/2-ERK1/2 [16]. Generally, activation of the MEK-ERK signaling pathway is a pro-survival signal, but activation of the MEK1/2-ERK1/2 pathway can also result in the induction of apoptosis [16]. Farnesol can induce phosphorylation of p65, which is dependent on activation of the MEK1/2-ERK1/2 signaling pathway in human lung adenocarcinoma cells (H460) [16], leading to increased p65 activity and chemokine ligand 3, IL-1, and COX-2 expression [16]. In addition, farnesol has shown to exert its pro-inflammatory effect by increasing the levels of IL-6, TNF-α, and COX-2 in macrophage cells (RAW 264.7) ( Table 2) [15]; these effects were markedly augmented when farnesol was combined with zymosan [15]. In an in vitro study carried out with primary murine splenocytes, farnesol treatment did not have an anti-inflammatory effect on the cells as it did not cause a significant change in the ratio of Th2/Th1 cytokines, as well as IL-10 and IL-2 production respectively, as compared to other sesquiterpenoid compounds [44].

In Vitro Anti-Inflammatory Effects of Farnesol
Apart from being produced by various plants, farnesol has also been excreted as a quorum sensing molecule by the fungus Candida albicans [45]. It also acts as a virulence factor, with mutants producing less and more farnesol becoming less and more infectious respectively [45]. The defense mechanism of the host organism in response to C. albicans infection can be usually mediated through the activation of acute inflammation, with Th1 cells producing pro-inflammatory cytokines to clear the infection [45]. Farnesol can function as a virulence factor by causing an anti-inflammatory response and suppressing pro-inflammatory cytokines, which makes the host organism more susceptible to the infection [45]. An investigation carried out with primary murine macrophages suggested that farnesol can also reduce the production of IL-12, which is a cytokine necessary for the differentiation of naïve T cells to Th1 cells as well as to stimulate the production of the pro-inflammatory factor, interferon gamma [45]. Therefore, farnesol may suppress immunity against C. albicans infection through the modulation of the inflammatory response.

Prostate Cancer
In diverse tumor cell lines, the phosphatidylinositol-3-kinase (PI3K) and serine/threonine kinase (Akt) signaling pathway is essential to regulate cell proliferation, cell survival, and apoptosis [46,47]. Activation of the PI3K and Akt signaling pathway inhibits the efficacy of chemotherapeutic drugs in various tumor cell lines [48]. Many reports have shown that inhibition of the PI3K and Akt

Prostate Cancer
In diverse tumor cell lines, the phosphatidylinositol-3-kinase (PI3K) and serine/threonine kinase (Akt) signaling pathway is essential to regulate cell proliferation, cell survival, and apoptosis [46,47]. Activation of the PI3K and Akt signaling pathway inhibits the efficacy of chemotherapeutic drugs in various tumor cell lines [48]. Many reports have shown that inhibition of the PI3K and Akt signaling pathway facilitates chemotherapy through the induction of apoptosis in prostate cancer cells [22,[49][50][51]. The MAPK family consists of three major members such as p38, ERK, and JNK, which respond to growth factors, cytokines, and stress to interfere with intracellular signaling associated with cell proliferation, cell death, cell survival, and transformation [52,53]. Farnesol-induced apoptosis in prostate DU145 cells was examined by Annexin V/propidium iodide staining [22]. Following treatment with farnesol, the protein levels of activated p-JNK, p-ERK, p-p38, p-Akt, and apoptosis-related signals including p53, Bcl-2, Bax, and cleaved caspase-3 were decreased [22]. Additionally, when LNCaP and PC-3 prostate cancer cells were treated with a farnesol and ibandronate combination, cell growth was inhibited; furthermore, farnesol alone appeared to be a potent inhibitor of tumor cell growth [23].

Breast Cancer
Duncan et al. reported that farnesol can induce the expression of thyroid hormone receptor (THR) β1, which inhibited cell growth in breast cancer cell lines [24]. Also, farnesol activated nuclear hormone receptors, such as farnesoid X receptor and peroxisome proliferator activated receptor-α/γ (PPARα, and PPARγ), which are steroid/thyroid nuclear receptor superfamily members that can regulate gene transcription [12,54,55]. In MCF-7 breast cancer cells, farnesol inhibited cell growth and induced THRβ1 protein/mRNA levels in a concentration-and time-dependent manner, but this effect was not observed in MDA-MB-231 breast cancer cells [24].

Lung Cancer
Using an XTT assay, it was found that farnesol reduced the cell viability of A549 and H460 lung cancer cells [25,27]. In the A549 cell line, farnesol treatment caused a cell cycle arrest of the cells in the G0/G1 phase, which subsequently resulted in apoptosis of the cells [26]. Since farnesol is structurally similar to the substrate of protein prenylation, farnesyl pyrophosphate, it was hypothesized that farnesol could act as a competitive inhibitor of prenyltransferases and inhibit the prenylation of Ha-Ras, a small G-protein, to suppress the activation of the Ras/Raf/ERK pathway [26]. However, it was demonstrated that farnesol-induced cell cycle arrest and apoptosis of the A549 cells were independent of the ERK pathway and farnesol did not affect the prenylation of Ha-Ras [26]. Alternatively, the anti-tumor effects of farnesol on the A549 cell line were due to its inhibition of the phosphatidylcholine (PC) biosynthesis pathway, where it suppressed the conversion of cytidine diphosphate-choline to PC, a step regulated by choline phosphate transferase (CPT) [26]. The substrate of CPT is diacylglycerol (DAG), and farnesol was also able to inhibit the activity of CPT by competitive inhibition with DAG. Addition of DAG rescued CPT inactivation by farnesol as well as preventing cell cycle arrest and apoptosis of the A549 cells [26]. In a previous study, farnesol was shown to induce the disorganization of the actin cytoskeleton in A549 cells, but addition of exogenous DAG and PC blocked its disorganization [26,58]. Moreover, supplementing the cells with exogenous PC was also able to attenuate the G0/G1 phase cell cycle arrest and apoptosis of farnesol-treated A549 cells, thus demonstrating that farnesol may also exert its anti-tumor effects through the regulation of PC biosynthesis [26].
In another lung cancer cell line, H460, farnesol treatment induced endoplasmic reticulum (ER) stress, which triggered the unfolded protein response (UPR) and caused apoptosis of the cells through the intrinsic apoptotic pathway, where there was an increase in the cleavage of caspase-9, caspase-3, and poly (ADP-ribose) polymerase (PARP), but not caspase-8 [27]. Farnesol treatment initiated the activation of the UPR signaling cascade, namely the PKR-like ER kinase (PERK)-eIF2α and the inositol requiring protein 1 (IRE1)-X-box binding protein 1 (XBP1) pathways [27]. This activation led to the upregulation of other UPR genes such as activating transcription factor 3 (ATF3) and DNA damage-inducible transcript 3 (DDIT3/CHOP) [27]. The induction of the UPR was also shown to be dependent on the activation of the MEK/ERK signaling pathway, since farnesol treatment augmented the phosphorylation of ERK, and the addition of a specific MEK inhibitor suppressed the induction of the UPR and inhibited apoptosis in response to farnesol [27]. Furthermore, it was shown that farnesol also increased the phosphorylation of other MAPK proteins, p38 and JNK, but the activation of p38 did not seem to play a part in the anti-tumor effects of farnesol [27]. On the other hand, inhibition of JNK did not block farnesol-induced UPR, but it did prevent apoptosis of the cells, suggesting that JNK may act downstream of the farnesol-induced MEK/ERK activation of the UPR to trigger apoptosis [27].

Pancreatic Cancer
Farnesol exerted its anti-tumor effects on pancreatic cancer cell lines, MIA PaCa2 and BxPC-3, by inducing a cell cycle arrest and triggering their apoptosis [28,30]. The cells were arrested in the G0/G1 phase of the cell cycle following treatment with farnesol, which was caused by the upregulation of p21 and p27 expression and the downregulation of cyclin A, cyclin B1, and cyclin-dependent kinase 2 (CDK2) [30]. In MIA PaCa2 cells, cyclin D1 expression was also reduced in response to farnesol [30]. In both cell lines, the increased levels of p21 and p27 resulted in augmentation of the p21/p27-CDK2 complex, which prevented the cells from progressing in the cell cycle [30]. BxPC-3 pancreatic cancer cells treated with farnesol had higher expression of Bak protein and an increase in apoptosis than non-treated cells [28]. In addition, MIA PaCa2 human pancreatic cancer cells and PC-1 hamster pancreatic cancer cells also demonstrated inhibition of cell growth following treatment with farnesol [29].

Cervical Cancer
In a study by Wang and colleagues, it was demonstrated that farnesol decreased the cell viability of cervical cancer HeLa cells in a dose-and time-dependent manner [31]. Farnesol also induced a loss in the mitochondrial membrane potential (MMP) and triggered apoptosis of the HeLa cells through the regulation of the PI3K/Akt pathway [31]. Farnesol downregulated the expression of PI3K and p-Akt proteins in HeLa cells in a dose-dependent manner to exert its anti-tumor effects [31].

Oral Squamous Cell Carcinoma (OSCC)
Farnesol was also able to exert its anti-neoplastic effects on OSCC cell lines, OSCC9 and OSCC25 [32]. Treatment with farnesol inhibited cell growth and induced apoptosis of the OSCC cells. There was an increase in the cleavage and activity of caspase-9 and caspase-3, as well as a decrease in the expression of the anti-apoptotic protein, survivin [32]. Furthermore, farnesol treatment upregulated the expression of several proteins involved in the suppression of carcinogenesis, such as Met-enkephalin, which is involved in the tumor immune function, and Mdm-2, a potent growth suppressor [32]. There was also a downregulation of several oncogenic proteins such as heat shock protein 27 kDa, RAN, and cofilin-1, which can function to inhibit apoptosis, induce genetic instability, cell cycle progression, and promote tumor metastasis [32].
It has previously been established that farnesol can inhibit the synthesis of PC, the precursor of DAG, a lipid second messenger [10]. The addition of exogenous DAG was shown to block the cell growth inhibition induced by farnesol as well as reduce the cleavage of caspase-9 and -3 to their basal levels. Therefore, it is likely that farnesol suppressed the production of DAG in OSCC cells to exert its anti-tumor effects [32].

Meningioma
Farnesol was also able to exert its cytotoxic effects on eleven primary meningioma cell lines of different World Health Organization grades and on the stable malignant meningioma IOMM-Lee cell line. The cell viability of the tumor cell lines was significantly decreased to below 10% after treatment with 1.2 µM farnesol for 24 h [33]. Farnesol also triggered apoptosis of the tumor cells in a dose-and time-dependent manner, where there was an increase and decrease in the expression of active and inactive caspase-3 respectively [33]. Single-stranded DNA was detected in the cells, signifying that apoptosis was taking place since the DNA was being denatured [33]. Additionally, IOMM-Lee cells displayed an augmentation in the level of cleaved PARP1, which is a substrate of caspase-3 and is cleaved during apoptosis [33]. Consequently, farnesol was able to inhibit the growth of meningioma by triggering apoptosis of the tumor cells.

Multiple Myeloma
In multiple myeloma U266 cells, farnesol inhibited the activation of transcription factor signal transducer and activator of transcription 3 (STAT3), a protein involved in tumor initiation as well as progression [34] and also downregulated cell proliferation, and the expression of anti-apoptotic related proteins [34]. Farnesol also reduced the expression of various proteins involved in cell proliferation, angiogenesis, and metastasis [56,57].

T Lymphoblastic Leukemia
The cell growth of lymphoblastic leukemia Molt4-hyg cells was inhibited and apoptosis was triggered when treated with farnesol. 75 µM farnesol increased the population of subG1 phase cells in a time-dependent manner, which was diminished upon the addition of the pan-caspase inhibitor, Z-VAD-fmk, to the treatment, indicating that farnesol dependent cell death may be mediated via caspases [35]. Moreover, it was found that farnesol upregulated the cleavage of caspase-9, caspase-3, and PARP, as well as induced a reduction of the MMP, thus increasing the expression of cytosolic cytochrome c in Molt4-hyg cells [35]. These effects were observed in a lesser extent in Molt-4 cells ectopically expressing Bcl-2, determining that farnesol-induced apoptosis of the T lymphoblastic leukemia cells was mediated through the intrinsic apoptotic pathway [35].
In Molt4-hyg cells, it was found that farnesol augmented the phosphorylation of eIF2α and the expression of several downstream unfolded protein response (UPR) genes, like activating transcription factor 3 (ATF3), DNA damage-inducible transcript 3 (DDIT3 or CHOP/GADD153), and homocysteine-inducible, ER stress-inducible, ubiquitin-like domain member 2 (HERPUD2) [35]. Farnesol caused downregulation of the UPR-related gene, GRP78, which can function as an anti-apoptotic protein to rescue the cells from ER stress [35]. In addition, farnesol treatment upregulated the phosphorylation of the MAPK proteins, ERK1/2, p38, and JNK [35]. Other studies have revealed that the UPR may be induced through the activation of the MAPK pathway, but in the case of farnesol-treated Molt4-hyg cells, the specific inhibition of the MAPK pathways did not result in a significant change in the expression of UPR genes compared to the control, signifying that farnesol-induction of the UPR may be independent of the MAPK pathway [27,35]. Thus, farnesol can exert its anti-tumor effects on Molt4-hyg cells partly through the induction of ER stress and activation of the UPR to trigger their apoptosis.

Anti-Inflammatory
Farnesol has been demonstrated to inhibit Ras protein and NF-κB activation and induce apoptosis in several in vivo studies (Table 3) [14,[17][18][19][20][21]. Farnesol significantly downregulated the Ras-Raf-ERK1/2 signaling pathway, which plays a key role in promoting cell survival, though this pathway can also play a role in growth inhibition and cell death [18,43,59]. Interestingly, a lower dose of farnesol was found to significantly inhibit the Ras-Raf-ERK1/2 signaling pathway, leading to the suppression of inflammatory gene expression, whereas a higher dose led to activation of the Ras-Raf-ERK1/2 signaling pathway in a skin tumorigenesis mouse model [18]. In addition, farnesol increased the Bax/Bcl-2 ratio, which led to induction of apoptosis in a skin tumorigenesis mouse model [18]. In asthmatic mice and gliosis-induced Swiss albino mice, farnesol supplementation decreased IL-6, TNF-α, and inducible nitric oxide synthase (iNOS), demonstrating that farnesol has an anti-inflammatory potential [14,21]. Other studies demonstrated that farnesol could provide lung protection by lowering the levels of lactate dehydrogenase in cigarette smoke extract-treated rats and B(a)P-induced pulmonary injury rats [17,20]. Moreover, other studies have reported decreased caspase-3 activity in 1,2-dimethylhydrazine induced colonic damage rats following treatment with farnesol [19].

Pancreatic Tumor
Farnesol can also abrogate tumor growth and/or reduce oxidative damage in various animal models in vivo [22,29,34]. In PC-1 pancreatic tumor xenograft hamster models, farnesol was added as a dietary supplement of 20 g/kg for 20 days [29]. Control group hamsters were fed a control diet for 20 days, and then compared with the farnesol fed group [29]. Farnesol attenuated tumor growth and the average tumor diameter of the farnesol treated cohort was significantly lower than that of the control on day 25 [29].

Prostate Tumor
Five-week-old male nude mice were injected subcutaneously with DU145 prostate cancer cells into their right flanks and divided randomly into two groups [22]. The farnesol treated group had 50 mg/kg oral treatment every day for 5 weeks [22]. After 29 days, the farnesol treated group demonstrated reduction in tumor growth, which was exemplified by smaller size and lower weight compared to the tumors in the control group [22]

Multiple Myeloma
Six-week-old female nude mice were injected with U266 multiple myeloma cells into the right flank and randomly divided into four groups [34]. The control group (group I) was treated with PBS 200 µL three times for 3 weeks, group II was treated with 60 mg/kg of farnesol three times for 20 days, group III was treated with the anti-cancer drug bortezomib 0.25 mg/kg once a week, and group IV was treated with farnesol (60 mg/kg) and bortezomib (0.25 mg/kg) in combination [34]. Each group was treated for 20 days by intraperitoneal injection, and tumor volume was measured for 5-days interval for 25-days [34]. Group II, III, and IV had significantly decreased tumor volume. Following excision, Immunohistochemistry showed that farnesol treatment reduced the levels of p-STAT3 and Ki-67, while western blot analysis indicated that it also suppressed STAT3 activation in order to induce apoptosis [34].

Conclusions and Future Perspectives
Overall, several studies have demonstrated the potential pro/anti-inflammatory and anti-cancer effects of farnesol in various diseases and cancers. Although farnesol displayed a more pro-inflammatory effect under in vitro settings, in vivo findings showed that it is likely to act in an anti-inflammatory fashion in various chronic inflammation-induced diseases such as asthma. This could be attributed to inflammation being a process that is dependent on the extracellular milieu, where various types of immune cells also contribute to a pro-or anti-inflammatory environment. Therefore, it is necessary to further investigate the in-depth mechanism of action of farnesol in modulating inflammation.
Additionally, studies have also revealed farnesol to be efficacious as a potential anti-cancer agent as it can exert cytotoxic effects against various neoplastic cell lines and it can significantly inhibit tumor growth in vivo. Farnesol can also regulate various pathways to exert its anti-cancer effects in tumor cells, which include the PC biosynthesis, MEK/ERK, UPR, JAK/STAT3, and PI3K/Akt signaling cascades. Since in most cancers multiple oncogenic pathways would be deregulated to contribute to the progression of the disease, farnesol could be beneficial as it can target different signal transduction pathways, making it a promising anti-cancer therapeutic. However, more research needs to be carried out to determine the safety and efficacy of farnesol for cancer treatment, which includes testing it in clinical trials, as its effectiveness, has not yet been exactly determined in cancer patients.

Conflicts of Interest:
The authors declare no conflict of interests.  50 Effective dose 50