Cloud computing supports many unprecedented cloud-based vehicular applications. To improve connectivity and bandwidth through programmable networking architectures, Software- Defined (SD) Vehicular Network (SDVN) is introduced. SDVN architecture enables vehicles to be equipped with SDN OpenFlow switch on which the routing rules are updated from a SDN OpenFlow controller. From SDVN, new vehicular architectures are introduced, for instance SD Vehicular Cloud (SDVC). In SDVC, vehicles are SDN devices that host virtualization technology for enabling deployment of cloud-based vehicular applications. In addition, the migration of Virtual Machines (VM) over SDVC challenges the performance of cloud-based vehicular applications due the highly mobility of vehicles. However, the current literature that discusses VM migration in SDVC is very limited. In this paper, we first analyze the evolution of computation and networking technologies of SDVC with a focus on its architecture within the cloud-based vehicular environment. Then, we discuss the potential cloud-based vehicular applications assisted by the SDVC along with its ability to manage several VM migration scenarios. Lastly, we provide a detailed comparison of existing frameworks in SDVC that integrate the VM migration approach and different emulators or simulators network used to evaluate VM frameworks’ use cases. Vehicular applications include safety, geo-advertising, and entertainment applications to make the vehicle’s passengers journey more enjoyable. The European Transport and Telecommunication Information (ETSI) defines Cooperative-Intelligent Transport System (C-ITS) applications that provide emergency (safety) messages and reduce drastically car accidents . In factThe Vehicular Cloud (VC) architecture ,4 is in The SDN is a growing networking concept in VANETs that brings better performance through Software-Defined (SD) Vehicular Network (SDVN). In SDVN, the vehicles and radio access at the RSU are considered as SDN devices (data plane). The SDN controller generates and maintains a database that includes network-wide knowledge such as network topology ,9. In adSDVN, Virtual Machine (VM) replication, and migration are joined together to improve the bandwidth of cloud-based vehicular applications. SDN-based Vehicular Cloud (SVC/SDVC) ,22 uses Firstly, we provide a comprehensive literature review on network and computation technologies for supporting SDN-based Vehicular Cloud (SDVC) such as VM migration, SDN, vehicular cloud computing, and cellular-to-everything (C-V2X) communication mode.Secondly, we provide an extensive description of SDVC in which the network forwarding devices such as vehicles and RSUs are decoupled from the control plane. The SDVC’s control plane is abstracted and managed by application functions available at either the edge or central cloud infrastructure.Thirdly, we outline and discuss potential cloud-based vehicular applications assisted by the SDVC. We further describe the ability of SDVC’s network control plane to manags several VM migration scenarios due to the high vehicle mobility in VC.Lastly, we provide a detailed comparison of existing frameworks in SDVC that integrate the VM migration approach, VM strategies, and different emulator/simulator networks used to evaluate VM frameworks’ use cases.In this paper, we discuss the working methodology of the state-of-the-art VM migration over SDN-enabled vehicular cloud computing integrated with MEC and VC. We also investigate the SDN-based vehicular cloud architecture by providing an extensive discussion of its functional system components to control the VM migration approach, mobility prediction, and heterogeneity of wireless interfaces equipped in vehicles. Then, we present the assistance of control layer to manage cloud-based vehicular applications on the basis of their ability to share computation, storage and bandwidth resources. Finally, an array of VM strategies and other design based VM migration techniques that require the attention of the researchers are presented. The design of VM migration modules to be executed into SDN controllers would allow predictable control of MEC forwarding devices during VM migration in SDVC. The main contributions of this article are as follows:The remainder of the paper is organized as follow. The background of technologies behind SDVC is detailed in In this section, a comprehensive review of the related work on the evolution of computing and network technologies for SDVC is presented. The section introduces: firstly, the VC; secondly, the vehicle-to-everything communication technologies; thirdly, the VM service in VC; and, finally, SDN and its concept for vehicular networks.Vehicular cloud: A local cloud that clusters a certain number of neighboring vehicles. An inter-vehicle network in an ad-hoc manner is established by Vehicle-to-Any (V2X) communications. The vehicles and everything in direct communication are considered as mobile cloud sites. They cooperatively create a vehicular cloud service that distributes computing and networking resources among the participating vehicles .MEC cloud: A edge cloud established among a set of distributed multi-access edge cloud. The MEC cloud provides a provision of various V2X-based cloud services through the distribution and integration of distributed edge cloud servers and cloud radio access networks according to the location of V2X users ,25.Central cloud: The traditional central cloud computing, which is established among a group of commodity servers on the Internet . A V2X uCloud-based vehicular applications and context-aware applications are offered to passengers in vehicles through cloud computing. To address the requirements for a proper Quality of Experience (QoE), the Vehicular Cloud Computing (VCC) thus opens up considerable new opportunities to execute context-aware applications without using resources of the traditional cloud infrastructure . VCC aimResources sharing in VCC enables computation, storage, and bandwidth as a service because of insufficient vehicle resources. In fact, sharing computing resources as a service, vehicles can offload compute-intensive tasks to the MEC cloud located at the proximity of the willing (consumer) vehicles. Indeed, cloud-based vehicular services are deployed as a VM. The replication and migration are managed either at the central cloud or at the edge cloud. On the other hand, VCC provides cloud-based vehicular service that requires shared storage as a service. Thus, a distributed storage at different edge computing infrastructures is a solution, e.g., for real-time on-board diagnostic (OBD) data . TherefoThe Third Generation Partnership Project (3GPP) through mobile broadband standardization has published LTE-V2X technology as a new standard for deploying V2X applications . The proA MEC cloud is accessible only by the nearby vehicles, i.e. those located within the radio coverage area. This fact helps us recall the concept of an RSU cloudlet . An RSU MEC computing should be able to keep cloud service by leveraging VM migration strategy. VM migration decision-making based enables MEC computation resources to keep the dedicated VM one hope distance from their owners (vehicles). These VM migration techniques reduce latencies and downtime when the VM resume at the next MEC infrastructure. For instance, Teka et al. proposed a VM migration strategy based on Multi-Path TCP (MPTCP) approach to keep the VM hosts using the same Internet Protocol (IP) to follow the movements of the users . EstabliThe basic logical structure of an SDN is shown in Communication between the control plane and forwarding plane in the SDN environment takes place through the SDN control protocol. In the SDN, because the control plane and forwarding plane are decoupled, a standard protocol with multivendor support was needed for communication between them. OpenFlow was developed for this purpose. OpenFlow was the first communication protocol that gives access to SDN controllers to program the rest of forwarding network devices . OpenFloThe SDN is an imperative technology to address existing challenges in vehicular networks. The control logic for existing networks is integrated into every underlying On-board Unit (OBU)  hardwareThe SDVN pioneers the concept of SDN in vehicular networks . The conThe ability to deploy an important number of vehicular applications through SDVN concepts brings the true meaning of the Intelligent Transportation System (ITS). Today, deployment of ITS applications demands higher agility in network restoration, massive scalability, faster deployment, and operating expenses optimization. ITS services simply cannot afford to be slowed down by the lack of speed in human-driven processes. Automation and programmable capabilities are needed to support the provisioning of ITS services, the monitoring of ITS networks, implementation of run-time changes due to the high mobility of vehicular networks and road traffic loads, etc. In addition, SDN offers a solution by linking ITS services to the vehicular network and reducing human intervention in the process of network management. The SDVN allows vehicular routing protocols to react to a dynamic environment and mobility of the vehicles. Furthermore, SDVN relieves the burden of open protocols to manage ITS applications. In fact, these open sources would run on top of the SDN controller through the northbound Application Programming Interface (APIs). Therefore, the features of SDVN would mitigate the issue of high mobility of vehicles, satisfy the requirements of ITS services, and enable heterogeneous radio access in the next generation vehicular networks.In this section, we provide an extensive description of SDVC and its related functional layers. The SDN architecture of SDVC is presented. We further discuss the assistance of SDVC’s control plane to support potential cloud-based vehicular applications.The architecture for deploying cloud-based vehicular network based on software-defined networking, MEC cloud, and central cloud computing is described in ,22. ClouFault tolerance of one SDN controller requires a hierarchical design with SDN-enabled core network, SDN-enabled computation resources, and SDN-enabled access network ,35. ThusThe MEC computing micro-data center includes the control layer and the forwarding layer. The control layer is abstracted in the computing hardware, which includes the host operating system, a hypervisor, and other software entities based on VMs, namely active VM controller(s), SDN communication infrastructure controller(s), and SDN OpenFlow controller. The active VM controller controls the next replication and migration of VMs. The SDN communication infrastructure controller has the role to select the wireless access communication radio to connect the central cloud or MEC network resources during VM migration and resume procedures. The SDN OpenFlow Controller is hereafter considered as the main SDN controller. This SDN OpenFlow controller controls the global management of the SDVC network as well as the active VM and SDN communication infrastructure controllers. SDN OpenFlow controller enforces the management of the SDVC, as shown in The forwarding layer includes forwarding devices such as an OpenFlow Wi-Fi access point and switch, local gateway, and local server service enablers. The VC supports V2X communications over C-V2X and Dedicated Short Range Communication (DSRC). The control plane decisions are not entirely made by a single SDN controller centralized element; adjacent SDN controllers in the MEC cloud could collaborate to make decisions. In fact, this collaborative decision is provided through mobility management, and Quality of Service (QoS) support provided at the SDN OpenFlow controller. The SDN OpenFlow controller should achieve a fast recovery after failures between adjacent controllers in the SDVC. A logically distributed control plane assisted by mobility management achieves optimum performance and robustness against failures .Information collection module of vehicles: The information collection module includes different types of sensors (safety and diagnostics sensors) in the vehicles. Availability of these sensors in the vehicle allows the information such as speed and revolutions per minute (RPM). Furthermore, surrounding or adjacent vehicles, pedestrians on the roads information, and weather conditions are collected for SDVC.V2X application module of vehicle: This module is considered to act as the traditional On-Board Unit (OBU) integrated in-vehicle wireless communication interface. It allows broadcasting information such as geo-messaging to MEC micro-data center to notify when the vehicles enter or exit certain regions of the roads. The V2X application of the vehicle will receive the message corresponding to the routing rules defined by the SDVC’s SDN OpenFlow controller. Moreover, the V2X application module receives and stores the current position information of the vehicle provided through the global position system (GPS) .Communication capable 3GPP LTE UE module of vehicle: The communication module includes the V2I, V2V, and V2N communications modules. V2I, V2V, and V2N communications should comply with both the requirements of LTE and service requirements for cloud-based V2X services. The V2V communication requires the communication module of a vehicle to get an exact subscription and authorization from a network operator. The communication module of the vehicle allows V2V applications to transmit no safety messages such as locations. The V2V communication module provides direct communication (LTE PC5) as it is defined under Release 12’s proximity services communications (“ProSe”) function . Radio cThe V2X access cloud includes vehicles and road users. Moreover, SDVC includes infrastructures to infrastructure links, a vehicle to network links, a vehicle to infrastructure links, a vehicle to vehicle links, vehicle to network links, and vehicle to vulnerable road users links. In addition, the V2X access cloud includes vehicles and UE-type RSU, which are considered as SDN devices similar to data planes in the SDN concept. Functions of the SDN devices in the V2X cloud are concentrated on data collection, forwarding, queuing, and sending data into the MEC micro-data center. Therefore, the vehicle-enabled V2X could be built with the following functional modules:Enhanced node BS (eNBs) type RSU deployment provides cellular communications to vehicles. In SDVC, UE-type RSU transmits wireless signals from or to the communication module of the vehicle through LTE-Uu. The communication module of UE-type RSU sends unicast messages to a MEC computing server. Furthermore, the information provided by the central cloud are distributed to all the vehicles into the relevant geographical area through either UE-type RSU or evolved Multimedia Broadcast Multicast Service (eMBMS).In general, SDN control in SDVC has the responsibility to harmonize the centralization concept of the SDN control with the native VC. A hierarchical control solution with a communication infrastructure controller and the active VM controller can be considered. The optimal placement of the controllers, routing overhead between them  challenge the management of VM migration service. In fact, the inter-controller communication throughput between the adjacent SDN controllers would be maximized through optimum algorithm pertaining to the main SDN controller’s placement with active VM and SDN communication infrastructure controllers.The SDN OpenFlow controller controls and forwards routing rules for, e.g., deploying any ITS services initiated from cloud computing. These rules are pushed to the SDN communication and infrastructure controllers through the northbound interface. The core architecture of the SDVC is adopted to logically centralize data forwarding and computation resources. The ETSI framework defines a reference for their deployment of V2X services that require ultra-low latency access . The accCloud-based vehicular applications rely on resource computation, storage, and bandwidth sharing to deliver services in terms of VM to the vehicles requesting cloud-based vehicular applications. A discussion of some cloud vehicular applications over the SDVC is given in the subsections below.Real-time navigation with computation resource sharing serves as a hypothetical use case to illustrate the potential benefits of applications of cloud-based vehicular networks . A vehicStorage resource sharing is an application where many data are stored on independent MEC storage resources. For example, video surveillance on a public transport bus uses high definition cameras to record real-time surveillance inside the public transport bus. This means the requirement of installing higher storage units. However, the cost of higher capacity units is not a suitable option. Cooperative storage is a valuable solution. The public transport bus accesses multiple MEC clouds toward its direction and stores the recorded real-time videos to its nearby MEC micro-data center’s server. The recorded videos are deleted either when the public transit bus finishes uploading them or when it drives towards the next MEC micro-data center. The SDN communication infrastructure controller provides the location of the next MEC micro-data center to record (store) recorded videos. SDN communication infrastructure controller determines the next location based on a set of information such as speed limit, available size of storage resource at the MEC micro-data center, and the access radio of the MEC micro-data center. With this set of information, the active VM controller would migrate the storage as service only to a MEC micro-data center that has enough storage space. The SDN OpenFlow controller updates routing path of MEC micro-data center that has in-vehicle video surveillance files. Content-based routing modules at the application layer would suggest the SDN OpenFlow controller to define routing rules so that the road operators would easily permanently retrieve at a given time a different slice of video stored on different MEC micro-data center. At the time of an accident, the road authorities would request the active VM controller to initiate routing rules to allow the transportation bus to upload the recent video not yet stored on the MEC micro-data center.Downloading from a central cloud a document by a passenger in vehicle is bandwidth consuming. In cloud vehicular network, a cooperative downloading is an option for allowing vehicles driving in the same direction to help other passengers download a large file. Vehicles in a group of V2X cloud download the file. For this scenario, a distributed active VM controller is established to control the cluster of vehicles downloading the file from the cloud. The SDN communication infrastructure controller relies on the vehicle collaboration sharing function (function at application plane) to update flow tables of the OpenFlow switch directly connected to vehicles with their vehicle Identification (ID). The vehicle collaboration sharing function enables bandwidth sharing application. In fact, this service accelerates the rate of network bandwidth by speeding up the download compared to one vehicle handling the download task. Thus, the active VM controller assists each vehicle (involved in downloading the file) to transmit its own part to the owner, which in turns merges (reassembles) all other downloaded parts to get the entire file.The management of cloud-based vehicular applications based on storage, computation, and bandwidth sharing are still worth discussing in the next generation vehicular network. Emergent technologies such as SDN and Network Function Virtualization (NFV) have potential to address the issue of ultra-low latency for cloud-based vehicular applications. Certainly, SDN and NFV would influence the deployment of cloud-based vehicular applications for the next generation vehicular network. This section provides the assistance of migration management of VM through the SDVC’s control plane. Four cases of vehicle mobility in VC are described. We further provide a review of existing frameworks of SD-based vehicular computing to assist VM migration approach. In addition, we discuss the state-of-the art of existing VM migration techniques. Furthermore, we present a comparison review that focuses mainly on the network emulators/simulators to evaluate SDVC’s use cases.The MEC cloud consists of the control layer and the forwarding layer. The assistance for VM migration is controlled through the control layer, which includes several SDN controllers. The SDN controller holds the management of the global network. It requires getting network status from SDN communication and active VM controller. The SDN communication infrastructure controller is in charge of adjacent MEC micro-data center and particularly manage the connectivity of heterogeneous radio access when the VM service resumes the adjacent MEC micro-data center. The migration of VM-overlay and VM-base is managed by the active VM controller as long as the vehicle moves from the current MEC micro-data center hosting the cloud-based vehicular services. The active VM controller manages the flow tables of MEC radio access and forwarding devices at the forwarding layer.The MEC micro-data center supports closest vehicles (V2X enabled UE). As the coverage of MEC micro-data center would experience short disconnection in case the vehicle moves far of the MEC radio access coverage. In , four scThe inter-MEC migration service concerns the VM migration from one MEC micro-data center to its immediate next MEC micro-data center when the two MEC computing resources are in different radio access network coverage. In this case, the active VM controller of MEC micro-data center-1 will not be migrated because the dedicated VM migrates to the next MEC micro-data center. Therefore, the active VM controller of MEC micro-datacenter would include routing paths to new packets from central cloud service. Then, the forwarding device of the radio access would take actions regarding the type of VM migration pre-defined in the in-coming packets field of flow entries. This scenario is shown on In the case of across MEC micro-data center computing, vehicles establish an ad-hoc network. In this case, the access radio coverage is the same for the two MEC micro-data center. During the trip, vehicle A accelerates at the point to break the ad-hoc connection. Vehicle C will access the radio access because vehicle D still hosts a VC with enough computing and network resources. The active VM migration on micro-data center-2 will then update routing rules to SDN devices on vehicle D so that vehicle C would continue hosting the dedicated VM of the current cloud-based vehicular service. In this scenario, the active VM in micro-data center-1 would continue control the routing rules of vehicle A. The SDN communication infrastructure controller would assume that vehicles D and C connect to the access radio deployed at MEC micro-data center-2. The SDN communication infrastructure controller in proximity would then allow the active VM controller-2 to establish forwarding rules to the rest of vehicles in the ad-hoc network established. This VM migration scenario is shown in The other case is shown in SDVC architectures integrate fog/edge computing to keep data and processing services as close as possible to the vehicular users but slight VM migration strategies, as presented in Cooperative driving services are enabled by architecture framework based on SDN, NFV, and MEC to support V2X network slices. In , distribService migration strategies based on SDN technology enhance delivery of cloud-based services to mobile users and vehicular users as well. Tarik et al. addresses user mobility and its related challenges by designing a migration strategy called Follow-Me Cloud . The FolThe SD-based vehicular cloud still lacks real simulation environment tools to better evaluate the performance of management of VM migration. The Mininet-WiFi  lacks clSimulator tools for the cloud network model based SDN controller are proposed in ,57. ThesThe lack of an SDN-based vehicular cloud simulator limits the performance of the control plane in the SDVN system. Fog computing addresses the issue of computation latency by leveraging service of VM migration assisted with migration policies. Strategies for service migration such as Follow-Me Edge, pre-copy based Live VM, and location-based VMM are implemented in ,62,69, rStrategies for service migration in no vehicular environments depend on mobility prediction solutions to enhance fog computing resources and global network traffic. MyiFogSim evaluates resources allocation in fog computing assisted by a user mobility prediction strategy while a VM service is migrating among fog servers towards users’s directions . MyiFogSVehicular cloud computing uses mobility prediction not only to allow service migration of VM running RSU/cloud services but also mitigate the challenge of high mobility and heterogeneity of wireless interface equipped in vehicles. Yao et al.  introducIn this paper, we summarize our findings along with lessons learned collected from our study on the state-of-the art accomplishment of SDVC-related cloud-vehicular applications and their VM management with other technologies  for offering cloud-based vehicular applications. Based on the many substantial research works we reviewed, academia and industry are proposing design and configurations of SDVC architectures that would support cloud-vehicular applications. Therefore, efficient resource management, low cost VM migrations and processing latency, are stringent requirements for delivering cloud-based applications. Although various VM migrations strategies have been proposed in vehicular cloud computing, the extensive investigation to evaluate the effectiveness of VM migration management of the SDVC remains an open issue. The researchers envision the use of SDVCs coupled with other technologies such as edge/fog computing and NFV. The control layer of SDVC would play an important role in the management of computing resources. In addition, research directions should focus on the implementation of intelligent selection algorithms to run inside the control layer of the SDN controllers in SDVC. For VM machine migration, when the vehicle decides to migrate the service, the MEC may require the SDN controller to choose the most suitable MEC server to start the service migration process. The SDN controller should be able to estimate the potential cost incurred and compare it against the resource utilization modules designed to improve the MEC cost and QoS from the point of view of end users. VM migration process in SDVC architecture can be further modeled using existing VM strategies discussed and implemented as applications that consider QoE requirements. The selection algorithms based on SDN concept would help to produce the best routing policy to decide whether to migrate a service or not. QoS-aware mobility multi-access selection algorithm; selection algorithm of migration path with both cost; resource allocation on multi-access edge servers; development of a high service migration mechanism for inter-MEC migration case, intra-MEC VM migration case, and micro-data computing across MEC; and central cloud to ensure service continuity are considered as research directions. In addition, an emulation platform for evaluating QoS and routing applications of SDVC applications is missing. The real traffic mobility using SUMO, for instance, might be included to evaluate the simulation of SD-based vehicular cloud applications. To support a range of unprecedented cloud-based vehicular applications that improve connectivity and bandwidth of cloud-based applications, SDVC is introduced. SDVC architecture considers vehicles to host virtualization services for enabling a VM to migrate from one RSU to another. In addition, we studied the evolution of computation and networking technologies of SDVC with a focus on its architecture within the cloud-based vehicular environment. Then, we discussed the potential cloud-based vehicular applications assisted by the SDVC along with its ability to manage multiple VM migrations. We also provided a detailed comparison of existing frameworks in SDVC that integrate the VM migration approach and different emulator or simulator networks used to evaluate VM frameworks’ use cases. In future work, we aim to provide further evaluation of discussed schemes based on their theoretical concepts and their performance. We will also consider the network resource slicing at the multi-access edge computing cloud for densely deployed vehicular network scenarios.


Of newly diagnosed HIV positive men who have sex with men (MSM) in the Netherlands, 29% have a non-Western migration background (MSM-NW). Among MSM-NW, HIV positivity rates are high (0.8%-2.0%), as is the proportion of late stage infections (39%). Factors such as HIV and sexual orientation–related stigma may form barriers for timely testing. Innovative approaches for HIV testing are needed to better reach MSM-NW. Social network testing (SNT) for HIV is an evidence-supported approach where peer recruiters identify persons (network associates) who could benefit from testing in their social or sexual networks. Web-supported SNT might be particularly promising for reaching people who may not be reached by regular care.The purpose of this paper is to describe the design of our pilot PREVENT (Peer-Empowered Voluntary Extended Network Testing). In this pilot, we will explore whether SNT using HIV self-tests is feasible and acceptable among MSM-NW in the Netherlands and whether it reaches those who were never or not recently tested for HIV (>1 year ago).The project aims to include 50 to 60 MSM and MSM-NW peers who will distribute 4 to 5 oral HIV self-tests each aiming to reach 200 network associates (NAs). Enrollment of peers includes 4 steps: (1) fostering interest in becoming a peer by health care professionals at sexual health clinics, HIV treatment clinics, and community settings; (2) sending peer contact information to the peer coordinator; (3) registering peers and giving program instructions by the peer coordinator and referring to the Web-based training at time2test; and (4) receiving precoded HIV self-tests for distribution in the peers’ networks. NAs who receive the self-test will log in with their test package code in the time2test application for step-by-step test instructions. After testing is complete, NAs receive tailored follow-up information depending on their test result.Between January and May 2019, 10 STI clinics and 7 HIV treatment clinics started recruiting peers. Results of the PREVENT pilot are expected in December 2020.This is the first Web-supported peer-driven SNT pilot using HIV self-tests in the Netherlands and one of the first in Europe. Implementation is considered successful if it reaches MSM-NW who were never or not recently tested for HIV. Additionally, it may encourage conversations within the networks about risk behavior and barriers to HIV testing, potentially contributing to the Joint United Nations Programme on HIV/AIDS goal of zero HIV infections.Netherlands Trial Registry NL7424; https://www.trialregister.nl/trial/7424DERR1-10.2196/14743 All participants in PREVENT provide online informed consent.The primary objective of PREVENT is to assess the feasibility and acceptability of SNT among MSM-NW, and therefore a formal power analysis is not appropriate. However, we aim to enroll 200 NAs during a period of 12 to 18 months. To achieve this target, an estimated 50 to 60 peers who will distribute 4 to 5 test packages each should be recruited when they attend any of the participating sites. With these numbers, the pilot is expected to have 80% statistical power to observe differences of 15% in study outcomes  between subgroups, such as younger versus older age groups or by migration background and location of recruitment.The e-learning tool is part of the time2test website that was developed for the PREVENT pilot. Findings of a qualitative prestudy among MSM-NW were used for website content and to design the e-learning tool . This quFrom the prestudy, learning objectives and delivery strategy were determined by the research team of PREVENT. Examples of learning objectives: peer can explain HIV transmission routes, peer can explain how the OraQuick HIV self-test  works, peer is motivated to prioritize friends within his inner circle of trust , peer is able to start a conversation about HIV testing. The learning objectives were translated into applications: the 6 steps of the e-learning tool in which they receive information and instruction videos on how to recruit NAs and motivate them to test. As there is no one-size-fits-all approach, example instruction videos were used. The prestudy showed that almost all men had an idea of how they would approach their contacts . Peers aIn step 1, peers watch an animated video about HIV and how it is transmitted; in step 2, the use of the OraQuick HIV self-test is demonstrated . In stepAt the end of the e-learning tool, the peer has learned the following:Explain what HIV is and how it is transmittedSelect NAs  for HIV self-testingMotivate NAs to test with an HIV self-testGuide NAs to the website for test instructions and counselingDeal with doubts or negative reactions of NAs about HIV testingAsk for support from the peer coordinatorNetwork associates who accept the self-test from their peers log in to the time2test website  with their test package number to create an anonymous test account based on a second code that is generated by the time2test application. The content of the time2test website as well as the instruction letters and manuals are provided in three languages .After reading about the aims, terms of participation, and data privacy, they give online informed consent for participation by clicking on a button “I have read and understood the information above.” Next, they are taken to the baseline questionnaire (12 questions) on demographics, sexual orientation, HIV testing history, and sexual behavior. After completing the questionnaire, participants watch an animation about the OraQuick HIV self-test and go to the step-by-step test instructions. The HIV self-test uses oral fluid from the gums to detect antibodies against HIV-1 and HIV-2. In an OraQuick HIV self-test usability study on 900 individuals, sensitivity was reported as 99.4%  and specificity as 99.0%  . The winNAs with a reactive (HIV positive) test are informed about the importance of having a confirmation test and preventing (possible) further transmission of HIV. They are referred for confirmatory testing to an STI clinic in their region (telephone numbers provided through the STI clinic finder) where they are given priority, or they can go to their general practitioner (GP). They can print or download a referral letter on their phone for the health care professional. The referral letter explains what HIV self-test was used and the need for confirmation testing. They can also bring the test manual from their test package. When the confirmation test is HIV positive, they are directly referred to the nearest HIV treatment clinic as part of standard practice.NAs with a nonreactive test are referred to the STI clinic, GP, or the Man tot Man (Testlab) website for repeated HIV testing after 3 months, testing for other STIs, and Web-based interventions aimed at reducing sexual risk behavior and increasing PrEP use.In case the self-test failed, NAs can ask for a new test via their peer or they can send an email to the project mailbox that will be answered by the peer coordinator. NAs who have questions about the test procedure are referred to the Frequently Asked Questions page on the website or they can contact a health care professional by telephone through the STI clinic finder on the website.Peers and NAs provide online informed consent for participation and data collection directly after the online project information page and prior to any online data collection. The baseline questionnaires inquire about age, migration background, educational level, sexual behavior (NAs only), and HIV testing history (NAs only) . After pAfter their SNT period, peers will be invited to participate in an evaluation survey and/or qualitative interview about their experiences with SNT, their role as a peer, and their relationship with the NAs they approached. We will examine factors supporting the acceptance of the social network approach, barriers that peers experience during the recruitment process, and outcomes on communication about HIV testing .NAs who used the self-test are asked to provide a voluntary email address in case they are willing to be contacted for a follow-up questionnaire after 3 weeks or become a peer recruiter themselves. The follow-up questionnaire contains questions about their experiences with SNT and the Web-based service for self-testing. Not giving consent to these options does not affect the ability to participate in the project in any way.The following outcomes will be examined (by study aim):Study feasibility and acceptability of SNT using HIV self-tests:Numbers of registered peers, peers finishing the training, peers starting SNTPeer profiles Network index (accepted/used tests divided by the number of peers)Numbers of NAs rejecting, accepting, and using the self-testNA profiles Types of relationships between peers and NAs Locations of self-test offerAssess the effectiveness of SNT regarding diagnosing HIV:Number of newly diagnosed HIV infections (self-reported and/or confirmed at STI clinic)Network yield (newly diagnosed HIV infections divided by the number of NAs/peers)NA characteristics associated with newly diagnosed HIV infectionsPeer characteristics associated with the ability to identify NAs with undiagnosed HIV infectionExplore the effect of SNT on openness toward HIV and HIV testing:Encouragement of conversations about risk behavior and HIV (testing) within networksReasons for nonacceptance of HIV self-testPositive or negative reactions by NAsPeer supportPeersInclusion:Demographics Approval to participate as a peerApproval for follow-up interviewFollow-up:Experiences with e-learning toolExperiences with supervision by the peer-coordinatorExperiences with distibution of HIV self-testsReactions to the test offerShared test experienceCommunication about HIV testing and sexualityWillingness to distribute more self-testsApproached network associates (completed by peers)Inclusion:Demographics Sexual preferenceType of network contactHIV testing historyHIV self-test acceptance and date of acceptanceReason for nonacceptance and date of acceptanceParticipating network associatesInclusion:Demographics Sexual behaviorHIV testing historyApproval for follow-up questionnaireFollow up:Reason for acceptance of HIV self-testExperience with the HIV self-test offerExperience with conducting the HIV self-testWould recommend the HIV self-test to othersHIV self-test conducted in presence of othersLocation of conducting the HIV self-testHIV test resultLocation of confirmation test (HIV positive self-test only)Result of confirmation test (HIV positive self-test only)Visited HIV treatment clinic (if confirmed HIV positive)Support by the peerSharing the test result with othersCommunication about HIV in social networkWillingness to become a peerWillingness to be interviewedChanges over time in online visitorsWebsite tracking dataInclusion:Frequency of log-insNumber of webpages accessedDescriptive analyses will be used to examine participation rates and characteristics of peers and NAs. The databases containing epidemiological data of peers and NAs can be linked based on the test package number. Therefore, we can identify person profiles  as well as social network profiles  and link these with SNT outcomes. Factors associated with (un)successful distribution of HIV self-tests will be examined by stepwise  regression analyses.Usability of the e-learning tool for peers will be evaluated by using mixed methods approaches  to gain insight on its user friendliness, record characteristics of peers (not) finishing the e-tool, and support enhancements in the (re)usability of the e-learning tool. Quantitative data will be analyzed using SAS/STAT version 14.1 (SAS Institute Inc) or SPSS Statistics version 24 (IBM Corp). Interview data will be transcribed using f4transcript version 7 (Audiotranskription) and assisted by NVivo software version 9  according to the thematic analysis method .Between January and May 2019, 10 STI clinics and 7 HIV treatment clinics throughout the Netherlands  began to recruit peers for the PREVENT pilot. A midterm and final evaluation will be conducted during 2019-2020.The PREVENT pilot is the first Dutch peer-moderated social network study using HIV self-tests and one of the first in Europe. This pilot intervention will provide insight on the feasibility and acceptability of SNT and its ability to identify undiagnosed HIV infections among MSM-NW in the Netherlands.A potential strength of this Web-based SNT approach is that hard-to-reach populations receive a free HIV self-test at a very low threshold, literally handed over by their friends. PREVENT has not only the ability to reach high-risk MSM-NW hidden to current HIV testing practices, it may also encourage conversations about risk behavior, HIV, and (barriers to) testing within the networks. Ultimately, the project can promote normalization of HIV testing. Also, the integrated online data collection, where peers and NAs complete questionnaires, enables anonymous linkage between the two groups. This provides us the opportunity to keep track of the distributed and used HIV self-tests.There are also challenges to the success of our implementation. The value of SNT depends on many contextual factors as shown in previous SNT studies -13. For During the evaluation of the pilot, we will focus on factors influencing all participation steps by peers and NAs, their feedback, and problem solving. We will also assess which peers reach the most NAs and who reaches NAs who have never tested before or tested positive. Factors include demographics, behavior, and process indicators. If needed, participation-enhancing strategies will be developed during the recruitment period. Creating general awareness for the pilot  is already considered to stimulate engagement of peers in SNT. The first results may also support the need for further improvement of the content on the time2test website, the integrated e-tool for peers, or other elements of our pilot implementation. If SNT is acceptable and feasible among MSM-NW in the Netherlands, it can easily be adopted by other clinics and/or the website can be adjusted for future recruitment in other HIV risk populations.


One of the major challenges in the treatment of cancer are differential responses of patients to existing standard of care anti-cancer drugs. These differential responses may, in part, be due to a diverse range of genomic, epigenomic, proteomic, and metabolic alterations among individuals suffering from the same type of cancer. Precision medicine is an emerging approach in cancer therapeutics that takes into account specific molecular alterations, environmental factors as well as lifestyle of individual patients. This approach allows clinicians and researchers to select or predict treatments that would most likely benefit the patient based on their individual tumor characteristics. One class of precision medicine tools are predictive, in vitro drug-response assays designed to test the sensitivity of patient tumor cells to existing or novel therapies. These assays have the potential to rapidly identify the most effective treatments for cancer patients and thus hold great promise in the field of precision medicine. In this review, we have highlighted several drug-response assays developed in ovarian cancer and discussed the current challenges and future prospects of these assays in the clinical management of this disease. Cancer is a major global health problem. According to the World Health Organization (WHO), cancer is the second leading cause of death behind cardiovascular disease, with one out of every six deaths being cancer-related. In the United States, the American Cancer Society estimates that in 2020 alone, there will be 1,806,590 new diagnoses and 606,520 cancer-related deaths [Traditional modalities of cancer therapy such as surgery, radiation therapy, and chemotherapy still remain the standard of care for clinical management of most cancers . AlthougCompanion diagnostics that have the ability to identify patients who are most likely to benefit from a particular anti-cancer therapy are being increasingly integrated with drug-development processes and clinical trials . HER2 ovDrug-response assays are in vitro platforms wherein live patient tumor cells are exposed to various chemotherapeutic and other agents in order to test their drug sensitivity. Development of such assays are specifically important in the context of ovarian cancer as most patients treated with the standard platinum-based chemotherapy eventually develop recurrent disease that is resistant to this treatment . Due to The majority of drug-response assays involve culturing primary patient tumor cells in a 2D or 3D cell culture environment where they are exposed to cytotoxic drugs . PerformChemo-response assays (CRAs) are ex vivo drug-response assays designed to characterize the sensitivity or resistance of a patient’s tumor cells to physician-selected, clinically applicable chemotherapy agents . Most CRTwo commercially tested CRAs in the US include Microculture-Kinetic (MiCK) assay  and ChemoFx assay . Both assays utilize 2D cell culture systems. The MiCK assay is based on the principles of drug-induced apoptosis in which neoplastic cells are purified from patients’ tumor samples, seeded in a 2D cell culture environment and exposed to serially diluted chemotherapeutic drugs causing apoptosis in tumor cells . The opt3 of tissue), it is accessible for testing core needle biopsy samples. Finally, this assay is highly automated allowing use of a wide range of drug concentrations. This assay has been utilized to predict the chemo-response of several types of solid tumors, including ovarian cancer [The ChemoFx assay is another 2D culture based ex vivo assay designed to predict therapy sensitivity or resistance of a patient’s tumor cells to a variety of chemotherapy drugs . After tn cancer ,19 and bn cancer . MultiplBeyond these two assays, there are several other 2D cell culture-based approaches that have been clinically evaluated in epithelial ovarian cancer as platforms for testing therapeutic responses. An overview of these assays is outlined in According to the American Society of Clinical Oncology (ASCO) guidelines (2011) the use of chemotherapy sensitivity and resistance assays is not recommended outside of the clinical trial setting . Most cuThe importance of interaction between cells and an extracellular matrix (ECM) was pioneered by Mina Bissell in the early 1980s where she and her team postulated that a reciprocal and dynamic interaction between cells and their surrounding ECM can modulate gene expression . Over tiOrganoids, resembling mini organ structures, are 3D multicellular aggregates grown in an ECM and utilized to model human organ development and disease in a dish ,33. OrgaSimilar to organoids, spheroids are compact 3D multicellular structures. Although these terms are often used interchangeably, they represent two distinct models of 3D culture. Spheroids are cultured under non-adherent conditions in serum-free media  and may Similar to spheroids, organoid 3D cultures can be utilized as tools for drug sensitivity assays because they also better recapitulate cell-cell interaction in a compact, self-organized structure compared to 2D cultures. Each model (spheroids vs. organoids) has its own advantages and disadvantages as reviewed by Gilazieva et al. . For exaTumor-derived organoids are patient-specific cancer organoids that are capable of partially preserving different tumor cell types as seen in vivo . These oTo fully utilize the potential of primary tumor-derived organoids as a platform for personalized medicine in cancer patients, scientists and drug companies are exploring the potential of large biobanks of organoids that may be used in predicting treatment response. Biobanks of patient-derived tumor organoids have been established for many tumors including colorectal , prostatOne short-term patient-derived tumor organoid study of high grade serous ovarian cancer was reported by Hill and colleagues wherein their research group established 33 organoid lines from 22 ovarian cancer patients . These oWith vast experience in ovarian cancer research, we have collaborated with colleagues and contributed to the development of a 3D organoid based platform for high throughput drug screening . In thisAs a drug testing platform, this approach offers several advantages . First, The tumor microenvironment (TME) plays a very important role in disease progression and metastasis . OvarianCo-culture based models are advanced cell culture techniques where tumor cells are grown together with cells such as fibroblasts or stromal cells that support their growth and development . Co-cultDynamic 3D models are another clinically relevant cell culture system capable of simulating the ovarian cancer TME. Some of these models utilize microfluidics to continuously feed cultured cells with fresh media and remove toxic waste products . CompareOne of the biggest challenges in cancer drug development is the selection of appropriate models for preclinical research. A preclinical model should ideally recapitulate the heterogeneity and genetic landscape of the tumor as closely as possible and should also be compatible with high throughput drug screening. The three main preclinical models for drug testing include cancer cells grown in 2D culture, organoids/spheroids grown in 3D culture, and in vivo patient-derived xenograft models. 3D cell culture models may emerge as the most promising tool in preclinical research as they combine two strengths: (i) the ability to recapitulate tumor architecture, and (ii) compatibility with timely high throughput drug screening.In this review we have highlighted the significance of chemotherapy-response assays in ovarian cancer. However, no ideal model (either 2D or 3D cell culture based) has been established or FDA-approved for clinical use yet. There are inherent challenges associated with developing these assays including (a) intra-tumoral heterogeneity, (b) inter-tumor heterogeneity between primary and metastatic sites from the same patient, and (c) absence of a tumor microenvironment. Intra-tumoral heterogeneity is known to play an important role in metastasis, invasion, therapy-resistance and tumor relapse . The exiIn summary, this article examines existing chemo-response assays and their potential in improving the clinical outcome of ovarian cancer patients by predicting therapeutic responses ex vivo. To be adopted for widespread clinical use, these assays should employ cell culture models that recapitulate actual tumor architecture, incorporate supporting tumor microenvironment cells for better modeling of disease in vitro, and be compatible with automation and high throughput analysis in a cost-effective manner. For clinical translation, it is also important that these assays are validated through well-designed prospective and blinded multi-center clinical trials. We believe that a reliable bioassay-directed selection of treatment not only could improve patient quality of life but also reduce the economic burden due to costs associated with administration of less effective treatment regimens. Hence, we hope that continued optimization of these assays may help provide an effective precision medicine approach for individualized treatment of cancer patients.


Heterologous immunity is defined as immunity that can be induced by a pathogen or antigen against another unrelated pathogen, antigen or even an autologous antigen. The applications of “heterologous immunity” can be traced back to the first vaccine by Edward Jenner in the late seventeenth century where he used a cowpox virus to immunize against smallpox infection. Following this, the concept of heterologous immunity and its implications for vaccine development and immunopathogenesis were largely neglected until the late 20th century. However, epidemiological studies involving various vaccinated populations continued to suggest the existence of heterologous immunity. With the availability of modern bioinformatics and immunological tools, heterologous immunity among several pathogens has now been recognized and the immunomodulatory role in protective immunity and immunopathogenesis is being studied extensively. The topic “Heterologous Immunity: Implications and Applications in Vaccines and Immunotherapies” covers current progress in the field of heterologous immunity and is a collection of 13 articles that includes reviews, minireviews and original research.Agrawal summarized the mechanism of heterologous immunity and its implications in pathogenesis and infectious disease outcomes. The mechanism of cross-reactive antibody or T cell immunity among related or un-related pathogens lies in the promiscuous nature of the interaction of B cell receptor and T cell receptor for epitope recognition. As a result, exposure to microbiota and various pathogens, and routine vaccination programs induce a considerable pool of cross-reactive T cells as well as antibodies able to respond to a completely different pathogen. The author further describes heterologous immunity among a broad range of pathogens and discusses its plausible role in natural resistance and modulation of the course of infection.The review article by Balz et al. outline the consequences of heterologous immunity in antiviral immunity. In this review, the authors highlight important applications of heterologous immunity in vaccine development. Accordingly, for influenza viruses, they suggest that future vaccine development efforts should be toward a universal vaccine focused on broader T cell responses. The authors further discuss the public health implications of heterologous immunity and suggest that more knowledge on heterologous immunity among various pathogens can provide insight into the repurposing of existing vaccines. They also point out factors that influence the clinical outcomes of heterologous immunity and specifically mention the benefits of measles and vaccinia vaccination in this regard.Rathore and St. John review cross-reactive antibody and T cell immunity directed toward Flaviviruses and their role in protection and the development of immunopathologies. The authors also detail rational vaccine design from the viewpoint of cross-reactive immunity.Flaviviruses are among the most common viral infections in tropical and subtropical countries. Heterologous immune responses among Flaviviruses are well-documented. Plasmodium parasite and is among the leading causes of death in endemic areas. In a comprehensive review, Mitran and Yanow describe cross-reactive antibody and T cell immunity among distinct species of Plasmodium that have been reported both in human populations and in experimental animal models. The authors define potential antigen targets for cross-species vaccine design in the context of heterologous immunity that would encompass evolutionarily conserved epitopes.Malaria, a mosquito-borne disease, is caused by the Tang et al. highlights this phenomenon in a study of infections in mice with mixed-species of malaria parasites. The authors show that co-infection of mice with Plasmodium yoelii with either Plasmodium vinckei or Plasmodium chabaudi increased virulence  compared to mono-infections where mortality was significantly lower . The authors discuss the role of immune competition/modulation on parasite virulence and the fitness costs of co-infection on transmission to mosquitoes.Heterologous immunity is particularly important from an immunopathology standpoint where it can negatively modulate the immune responses against another pathogen. This can occur through the induction of biased non-protective cross-reactive antibodies and T cells. The research article by Youssef et al. identified a monoclonal IgM antibody against fungus Candida albicans' Hyr1 antigen that cross-reacts with the Gram-negative bacteria Acinetobacter baumannii and Klebsiella pneumoniae. Moreover, this antibody protects mice from lethal bacteremia. This study shows that a single monoclonal antibody can provide cross-kingdom protection against multiple microorganisms.Apart from vaccines, heterologous immunity is an attractive approach for the development of immunotherapeutic monoclonal antibodies. Rowntree et al. published an elegant study where CD8+ T cells specific for different viral epitopes  cross-reacted with HLA-B27 allotypes in a hierarchical manner. This study clearly shows that heterologous immunity has clinical implications wherein antiviral T cells induced against viral pathogens could contribute to adverse outcomes in allogeneic transplantation. It also implies that exposure of a host to multiple viral infections could lead to a more complex allo-reactivity.The T cell receptor has remarkable promiscuity toward its antigen epitopes and this feature is responsible for the generation of cross-reactive T cells. Cross-reactive T cells have several clinical implications for immunopathogenesis, autoimmunity, and alloreactivity and graft rejection. For example, Soon et al. shows that a therapeutic T cell specific to a hepatitis E virus epitope cross-recognized an epitope from an apoptosis-related autoantigen in the host, which could potentially lead to autoimmunity. Most importantly, the authors characterized a molecular signature of a multiple-glycine motif in the CDR3 region of the TCR β chain which may allow greater structural flexibility with a minimum energy threshold required for promiscuity. Therefore, such T cell-based therapies should be evaluated for any off-target cross-reactivity.The advent of newer T cell-based therapies (such as CAR-T cells) and their widespread clinical uses can be vulnerable to the cross-reactive nature of the T cell receptors. A study by in silico and immunological tools to design cross-reactive, conserved antigen epitopes as well as the use of a suitable adjuvant formulation that favors the induction of antibodies and T cells with broader specificities. Nguyen et al. show that the Pandemic H1N1 vaccine formulated with poly-γ-glutamic acid (PGA)/Alum complex provided cross-protection against heterologous influenza viral strains: A/Puerto Rico/8/34 (H1N1) and A/Hong Kong/1/1968 (H3N2)]. In a similar study, Luo et al. showed that the H7N9 vaccine formulated with STING agonist cGAMP could provide effective cross-protection against H1N1, H3N2, and H9N2 influenza viruses in mice. Finally, Lee et al. showed that mincle and STING-stimulating adjuvant formulated with a foot and mouth disease virus vaccine induced a robust and long-lasting cellular and humoral memory response across diverse species in mice, cattle and pigs. These studies suggest that the use of new, advanced adjuvant formulations will not only induce antibodies and T cells with broader specificities for greatly improved immunity but also overcome disparities in immunogenicity of a vaccine across species.Heterologous immunity is also relevant for vaccine development against highly diverse pathogens where cross-strain reactivity or cross-species protection is critically desirable. As an example, current vaccine development efforts against influenza viruses are focused on developing a broadly cross-reactive universal vaccine. These efforts employ modern Covián et al. reviewed BCG vaccine-induced trained innate immunity and its role in cross-protection and heterologous effects.Heterologous immunity is not only limited to adaptive immunity. Innate immunity triggered by one pathogen or vaccine can protect against an unrelated pathogen. This is often called “trained immunity” and is represented by innate immune cells. Trained immunity arises due to epigenetic reprogramming of innate immune cells such as macrophages upon exposure to infection/vaccine. Connolly and Hussell review several aspects of this training of macrophages and the influence of Type 1 interferons on the alveolar macrophage. The authors also discuss how influenza virus-mediated production of Type 1 interferon alters the functional response of macrophages toward bacterial superinfection.Furthermore, macrophages play a distinct role in heterologous immunity because they have different functional stages at various points in the course of an infection. This process results in an intrinsic functional imprinting/training of macrophages by the invading pathogen. Collectively, these reviews and original research articles provide a comprehensive overview of the various dimensions of heterologous immunity. They support the outlook that heterologous immunity should be an important aspect in the design, testing and development of vaccines and immunotherapeutics. In addition, the influence of heterologous immunity should be carefully examined on clinical outcomes of infections and autoimmunity.SS wrote the manuscript. BA and SY performed critical revision and editing. All authors share equal intellectual contribution to this work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, the use of natural killer (NK) cell-based immunotherapy has shown promise against various cancer types. To some extent therapeutic potential of NK cell-based immunotherapy depends on migration of NK cells towards tumors in animal models or human subjects and subsequent infiltration. Constant improvement in the pharmacological and therapeutic properties of NK cells is driving the performance and use of NK cell-based immunotherapies. In this review, we summarize the molecular imaging techniques used in monitoring the migration and infiltration of NK cells in vivo at preclinical and clinical levels. A review of pros and cons of each molecular imaging modality is done. Finally, we provide our perception of the usefulness of molecular imaging approaches for in vivo monitoring of NK cells in preclinical and clinical scenarios. Natural killer (NK) cells, a type of lymphocyte, were first reported almost four decades ago. They are granular in nature and are involved in immune-surveillance . NK cellOncologists have questioned the tumor homing capability of ex vivo expanded NK cells, and their cytotoxicity in the tumor microenvironment. Over the past 20 years, only a few clonal NK-cell lines had been established . The NK-92 line was derived from peripheral blood of 50-year old Caucasian man with non-Hodgkin’s lymphoma . NK-92 cRealizing the therapeutic potential of NK cells requires more knowledge of cell characteristics. Non-invasive in vivo real-time imaging of NK cells in animal tumor models and human subjects become important in this context to determine the NK treatment success and efficacy. In this review paper, we discuss molecular imaging techniques used to evaluate the migration and infiltration of NK cells. We also discuss the advantages and shortcomings of approach. Finally, we review the development of the technologies and designs that has led to the currently available sophisticated methods with specific in vivo examples.Precise in vivo imaging of NK cell migration is extremely useful for basic and advanced biomedical researches, and for practical applications. Optical imaging methods are especially well-suited for visualizing infused cells in preclinical animal studies as they are very sensitive and non-invasive ,19. OptiAdvantages of nuclear imaging using radionuclides are excellent sensitivity and no depth limitation . CompareTwo is better than one, so dual or hybrid imaging using nuclear imaging combined with anatomical imaging, such as computed tomography (CT) or MRI, are clinically available. Therefore, allowing us to examine the functional and also structural imaging at the same time. The hybrid technology allows accurate localization of the NK cells in subjects; MRI with an advantage of excellent resolution for anatomical structures has been successful in this regard as nuclear imaging has low anatomical resolution (PET/SPECT) ,33,34. ADevelopment of NK cell-based cytotherapy requires a clear understanding of migration followed by the infiltration of NK cells to tumors and an ability to quantitatively analyze their in vivo fate after administration. Noninvasive in vivo imaging modalities for the administered NK cells might permit the comprehensive understanding of the in vivo therapeutic effects of NK cells for diseases including cancerous diseases by providing accurate data of the in vivo distribution and kinetics of the cells. Recent advancements in in vivo molecular imaging permit the recognition of simultaneous multiple biological processes within living organisms . Transla3O4.SiO2 core/shell nanoparticles to control the migration of the NK cells in the external magnetic field. Fluorescence imaging revealed migration control of NK cells by the technique. The NK cells loaded with nanoparticles migrated to the tumor and infiltrated into the tumor up to 17-fold greater compared to the control. The labeling procedure did not affect the killing activity of the NK cells [Lim et al. studied the in vivo cytotoxicity effects of NK92MI cells labeled with anti-human CD56 antibody coated QD705 (near infrared [NIR]-emitting fluorescent quantum dot) using melanoma tumor-bearing mice. NK92MI cells were retained in the tumors 24 h following intratumor injection as shown tumor by fluorescence imaging. Localization of NK cells in the tumor is one of requisites for antitumor activity. The observed reduction in tumor size suggested the potential therapeutic effect of the administered NK cells. Viability and therapeutic effect of the NK cells were not influenced by the fluorescence imaging agent to NK cells . HoweverNK cells . BiolumiNK cells  but inveNK cells .Wennerberg et al. demonstrated NK cell migration using a NIR lipophilic dye (DiR) in a xenograft mouse model. The study was based on the knowledge that NK cells express chemokine receptor chemokine receptor 3 (CXCR3), the receptor for CXC ligand 10 (CXCL10). To exploit this chemoattraction, the authors transfected a human melanoma cell line with CXCL10 then implanted the cells into mice. Fluorescence imaging revealed that intravenously injected DiR-labeled NK cells migrated and infiltrated to melanoma expressing CXCL10 rather than to negative melanoma tumor . Tavri eZhu et al. transduced the firefly luciferase (Fluc) gene into NK cells (NK/Fluc) and used bioluminescent imaging to track the migration of intravenously injected cells towards the anaplastic thyroid cancer (CAL-62) in mouse models with lung metastasis or xenograft. Imaging revealed that NK/Fluc cells migrate and infiltrate into the lung tumor within 1 h, remain there for up to 24 h and then reduced in number at 48 h. Furthermore, in a xenograft model, intravenously injected NK/Fluc cells migrated to the lung within 1 h and then migrated to the xenograft tumor within 3 h. The cells remained in the tumor for up to 24 h, with reduced cell number detected at 48 h . OpticalOther researchers investigated the migration of NK-92-scFv(MOC31)-zeta cells to EpCAM-positive prostate cancers in a rat model using MRI. Ferumoxide was used for labeling to permit their tracking. These cells and ferumoxide-labeled EpCAM-targeting NK-92-scFv(MOC31)-zeta cells were intraperitoneally injected in a prostate tumor-bearing rat. Imaging demonstrated marked negative tumor enhancement 1- and 24-h post-injection of the labeled NK cells. The signal remained unchanged at 1- or 24-h post-injection of the labeled NK-92 cells. The observations clearly indicated that NK-92-scFv(MOC31)-zeta cells migrated to the tumor site killed tumor cells . One fla19F perfluorocarbon and assessing their tumor infiltration in neuroblastoma mouse model. MRI revealed cell infiltration into tumors at day 0 and 2. The labeled cells were also injected via the same route into mice harboring human mantle cell lymphoma. MRI revealed tumor infiltration of the cells at days 0, 1, 3 and 8. Upon subcutaneous injection of labeled cells into melanoma-bearing mice, tumor infiltration was observed on days 0, 1, 3, 7, 10 and 15  methyl iodine and some of them were activated by interleukin (IL)-2. Activated and non-activated NK cells were injected into the lateral tail vein distal to the fibrosarcoma. PET imaging revealed no change in the migration of activated and non-activated NK cells at 30 min, while at 60 min a stronger tumor signal was obtained with the activated cells. PET imaging visualized accumulation of activated NK cells localized in small tumors fluorodeoxyglucose (FDG)-labeled genetically modified NK-92-scFv(FRP5)-zeta cells to HER2/neu-positive sarcoma in a mouse model. Tumor migration of the genetically modified cells was found to be two-fold higher compared to parental NK cells. Labeling efficiency was 81% after 60 min of incubation and cell viability was unaffected by the labeling protocol. However, in order to carry out NK cell monitoring for a longer duration the strategy should be selected [99mTc-anti-CD56 monoclonal antibody. NK cells accumulated in anaplastic thyroid cancer within 24 h post-intravenous injection [In another study, autoradiography was used to assess the migration of [selected . Finallynjection . Nuclearnjection ,35,48. MIn the above reviewed studies, nine of the studies used the primary NK cells, followed by five studies with NK-92MI and four studies with NK-92. In addition, KHYG-1, LNK and murine NK cells were used by one study each . GeneralSince each modality has its own limitations, multimodal hybrid imaging consisting of two or more imaging technologies can provide more informative results. Commercial systems integrating optical, PET, SPECT, CT and MR technologies in various combinations are already available. These hybrid techniques allow integration of the strengths of individual modalities. For example, recent advancement in tomographic fluorescence fusion systems allowed us to combine optimal imaging and high-resolution anatomical imaging techniques such as CT or MRI. This way, we can overcome the flaws in optical imaging approaches such as poor resolution. Likewise, a hybrid of PET and MRI can offer high sensitivity of PET and excellent resolution of MRI together, which are clinically relevant characteristics. Therefore, the hybrid imaging can be very useful for in vivo monitoring of NK cells in patients. These emerging developments can accelerate clinical application of promising NK cell-based therapies by accurate in vivo monitoring of administered therapeutic NK cells. Better in vivo NK cell monitoring should be possible with advancement of tracer chemistry and imaging technologies, which will ultimately progress the field of NK cell-based immunotherapy with further improvements in our understanding of genetic regulation in the NK cells.Successful NK cell migration and infiltration relies on a variety of factors to ensure that NK cells reach the tumor and are able to eliminate it without being suppressed in the process. Continued studies on the intrinsic mechanisms of NK cell biology and regulation are warranted to develop more safe and efficient therapeutic strategies. Our knowledge of how NK cells migrate to tumors and other components of the immune system will continue to increase with the help of various molecular imaging technologies. This knowledge will contribute to the selection of the most suitable NK cell-based immunotherapy for certain clinical scenarios. Our capacity to design more effective personalized NK cell therapies will likely increase in the near future.


Participants were randomized  to one of two sequences; consumption of fish fed with fish oil diet /fish fed with olive pomace-enriched diet  versus EF/CF. The primary outcomes were ex vivo human platelet aggregation and circulating plasminogen activator inhibitor-1 (PAI-1) and P-selectin (sP-selectin) concentrations. EF consumption had no significant effect on platelet sensitivity or haemostatic markers compared to CF. Platelet sensitivity to platelet-activating factor (PAF) decreased after CF consumption during the second period (p < 0.01). Plasma PAI-1 and sP-selectin concentrations increased after CF consumption during both periods (p < 0.01 for both). Based on current findings, consumption of enriched farmed gilthead sea bream had no greater effect on coagulation markers in adults compared to the conventionally fed fish.Fish consumption beneficially affects coagulation markers. Few dietary intervention studies have investigated differently fed farmed fish against these cardio-metabolic risk factors in humans. This double-blind randomized crossover trial evaluated differently fed farmed gilthead sea bream consumption against platelet aggregation and circulating haemostatic markers among apparently healthy adults. Subjects aged 30–65 years, with a body mass index 24.0–31.0 kg/m Fish is an integral part of a diet aiming to prevent or treat cardiovascular disease (CVD). Governing bodies worldwide currently recommend consumption of fish one to three times per week, with great emphasis being placed on the consumption of fatty fish to increase the dietary intake of long-chain ω-3 polyunsaturated fatty acids (PUFAs), i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) . In healsn-glycerol-3-phosphocholine) [Platelets play a critical role in the physiological process of haemostasis and also in the formation of pathological thrombi ,6. Platecholine)  is the mcholine) . Subclincholine) ,12. Dietcholine) . Howevercholine) . In a rePart of the ongoing aquaculture research has focused on plant-based products as substitutes for raw materials of marine origin, and although many studies have examined the effects of different fish feeds on fish growth, performance and mortality, few studies have addressed the potential benefit of modifying fish feed for human health . Specifisn-2-acetyl glycolipid, was found to stabilize established atheromatous lesions in animal models of atherosclerosis, and these effects were comparable to a standard chow diet supplemented with simvastatin [Several PAF-inhibitors of natural origin have been identified, which can either bind to the PAF-R or inhibit certain processes in the PAF-induced signal transduction pathways . Among tvastatin . Nasopouvastatin .The aim of the study presented herein was to evaluate the effects of consumption of fillets from differently fed farmed gilthead sea bream on platelet aggregation and circulating haemostatic markers among apparently healthy adults. Based on the findings of the in vitro experiments on the inhibition of the PAF-induced aggregation , we hypoThe trial was a double-blind randomized dietary intervention study with a 1:1 allocation ratio, conducted in accordance with the Declaration of Helsinki . This st2 who met the eligibility criteria for habitual fish consumption (<150 g of cooked fish per week). Exclusion criteria were pregnancy, current or recent weight loss effort, use of dietary supplements and being under treatment for any medical disorder. Subjects were allowed to use medical treatment for thyroid gland disorders, iron or folic acid supplements, contraceptives or hormone replacement therapy (HRT) for women, provided they would continue receiving their medication throughout the study. Eligible subjects were recruited from the community by word of mouth, and were all living in the Attica area, Greece. The study was carried out at the Laboratory of Nutrition and Dietetics in collaboration with the Laboratory of Biology, Biochemistry, Physiology and Microbiology of Harokopio University, Athens, Greece.Eligible subjects were all adults aged between 30 and 65 years old with a body mass index (BMI) between 24.0 and 31.0 kg/mSparus aurata L.), and the two types of fish compared were farmed fish fed with fish oil diet (CF) and farmed fish fed with olive pomace enriched diet (EF). NIREUS Aquaculture S.A. provided both types of fish. The production method of fish feed enriched with olive processing byproducts, the method to enrich farmed fish and the effect of feed on the fatty acid profile and total polar lipid (TPL) concentration in the flesh of gilthead sea bream, are described in detail elsewhere [within-group > 0.5, for all). During the 6-week washout period, participants were not provided with any fish portion and were advised to continue their long-standing dietary habits.Participants were randomly assigned to one of two fish treatment sequences (CF/EF or EF/CF). Participants in each sequence received each treatment for 8 weeks with a 6-week washout period between treatment periods. The fish species studied in the present trial was gilthead seabream , and ex vivo assessment of human platelet aggregation in platelet rich plasma (PRP). All assessments were performed at the beginning and at the end of each treatment period.2). Waist circumference was measured to the nearest 0.1 cm between the lowest rib and the superior border of the iliac crest at the end of normal expiration. The American Heart Association/National Heart, Lung, and Blood Institute criteria were used to define increased waist circumference among study subjects . SBP and DBP of subjects were measured twice, 2 min apart, in a sitting position, using an automated Omron M4 sphygmomanometer and the average of the two measurements was calculated and recorded.Anthropometric assessment: Body weight of participants was measured with digital scale  to the nearest 100 g, and body height was measured to the nearest 0.5 cm. BMI was calculated as weight (kg) divided by height squared  validated for the Greek population . This quSmoking assessment: Subjects were assessed with respect to smoking status , duration of smoking (in years) and mean number of cigarettes smoked per day (for current smokers). Pack-years of smoking were calculated based on the following formula:® Plus Plastic Serum Tubes). Blood was allowed to clot at room temperature for 60 min until centrifugation . Plasma was obtained from EDTA tubes (BD Vacutainer® spray-coated K2EDTA Tubes). Whole blood count was performed on a BC-3000 Plus Auto Hematology Analyzer  at room temperature immediately prior to plasma collection. All serum and plasma aliquots were stored at −80 °C until biochemical analysis.Blood sampling: The subjects were asked not to use any non-steroidal anti-inflammatory medication, antibiotics or analgesics two weeks before the scheduled time of blood collection. The subjects were also instructed to avoid vigorous physical activity the day before blood sampling and refrain from smoking in the morning prior to blood collection. Venous blood samples were drawn after a 10-h overnight fast between 08:00 AM and 10:00 AM. Serum was obtained from silicone coated tubes  based on enzymatic methods were used to determine glucose (mmol/L)  2.4%), triacylglycerols (TAG) (mmol/L) , total cholesterol (mmol/L) . High density lipoprotein-cholesterol (mmol/L) was determined using the same method as that used for total cholesterol, after precipitation of non-HDL lipoproteins with phosphotungstic acid and LDL-cholesterol (mmol/L) was calculated using Friedewald formula. Insulin (pmol/L) was measured with an enzyme-linked immunosorbent assay . The homeostasis model assessment (HOMA) method was used to assess insulin resistance (IR) from basal (fasting) glucose and insulin concentrations; HOMA-IR was calculated according to the formula: (FPI × FPG)/22.5, where FPI is fasting plasma insulin concentration (mU/L) and FPG is fasting plasma glucose (mmol/L). The concentrations of PAI-1 (pg/mL) and sP-selectin (ng/mL) were measured in duplicate in plasma. Plasma concentration of PAI-1 was measured with an enzyme-linked immunosorbent assay . Plasma concentration of sP-selectin was measured with an enzyme-linked immunosorbent assay .50 or half maximal effective concentration) can be calculated from the linear part of the aggregation curve. As expected, the lower the EC50 value for an agonist the higher the potency of its platelet aggregation effect. Bovine serum albumin (BSA), PAF, ADP and THR were obtained from Sigma-Aldrich . EC50 measurements were expressed in mM (for ADP and PAF) or in U/mL (for THR).Ex vivo assessment of human platelet aggregation: The procedure followed has been previously described . Briefly50 values of platelet aggregation induced by PAF.A priori power analysis showed that a sample size of 25 participants was adequate to achieve statistical power equal to 83% at a 5% significance level of two-sided hypotheses that evaluated 1 standard deviation (SD) difference between groups based on the ECParticipants were randomly allocated to each sequence group using a computerized random number generator. One of the research dietitians was solely responsible for the assignment of participants to the treatment sequences, whereas all other team investigators were kept blinded to the allocation. In addition, the participants were not aware of the fish treatments provided since there was no obvious difference between the two types of raw fish, and the enriched cooked fish had similar sensory characteristics  to the conventional one .During the fish consumption and the washout periods, participants were asked to record the days they consumed fish, and the use of any medication, as well as to report any short-term cold. All records were collected during the appointments that participants had with the research dietitian. Moreover, in the middle of each intervention period, one of the investigators called participants to explore compliance to the intervention and to record any unexpected conditions . In addition, participants’ compliance with the study protocol was assessed with a fatty acid analysis (FA) of red blood cell (RBC) membranes collected at the beginning and the end of each fish treatment period.® spray-coated K2EDTA Tubes) after plasma collection. Fatty acid methyl esters (FAME) were produced during methanolysis of the RBC extracts, and were extracted with hexane containing internal standard, i.e., methyl nonanoate, and BHT. Fatty acid methyl esters were analyzed with gas chromatography using an Agilent HP-6890  gas chromatograph equipped with flame ionization detector, split–splitless injector, and an HP 6890 autosampler. A standard FAME mixture was used for the identification of the peaks . For the purposes of the present study, only those FAs that comprised >0.01% of the total FAs of the RBC membranes were considered for data analysis, i.e., 34 FAs out of a total of 46 FAs identified. Trans isomers were not included.Red blood cell isolation and FA analysis: The procedure followed has been recently described . BrieflyAll statistical analyses were performed using Stata Statistical Software, Release 12 . The significance (α) level was set to 0.05 for all tests. The Shapiro–Wilk test and normal Q–Q plots were used to test normal distribution of data. Data were analysed based on the per-protocol principle.t-test or the nonparametric Mann–Whitney test were used for variables following a normal or a non-normal distribution, respectively. Categorical variables  are presented as frequencies, n (%); relative frequency (%) is expressed as percentage of all participants (n = 30). Continuous variables with normal distribution are presented as mean ± standard deviation (SD), whereas continuous variables with non-normal distribution are presented as median . p-values are presented for two-sided two-independent-samples tests.The two sequence groups (CF/EF vs. EF/CF) were compared for demographic, anthropometric, biochemical, physical activity and dietary characteristics assessed at the beginning of the study. Fisher’s exact test was used to assess if sex and smoking status were uniformly represented between the two groups. For continuous variables, two-independent-samples t tests were used to evaluate whether the RBC membrane content in EPA and DHA was higher after fish consumption compared to the content before fish consumption. Comparisons were performed by period and treatment. Data are presented as mean before treatment ± SD, mean change ± SD, 95% confidence interval (CI) of the change, and p-value for one-sided paired-samples t tests.Paired-samples For each primary outcome and other outcome measure, sequence, carry-over and period effects were evaluated with analysis of variance for a 2 × 2 crossover design (pkcross), and treatment effect was estimated with linear regression (regress). Input data were expressed as relative change (%), calculated as the change between values at the end and values at the beginning of each treatment period. For data with non-normal distribution, the natural logarithm (ln) of the ratio (%) was used instead of the relative change (%). Linear regression models were fitted for each of the outcomes. Treatment was used as the main exposure, and the CF was used as the reference group. Adjustments were made for the period of treatment, i.e., 1st period (winter) versus 2nd period (summer). Additional adjustments for insulin or HOMA-IR, and pack-years of smoking did not alter the results and thus are not presented. The effect of treatment is presented as the exponentiated coefficient  and 95% CI.t tests or Wilcoxon signed-rank tests were used for this analysis. p-values are presented for two-sided paired-samples tests.For each primary outcome and other outcome measure, values at the beginning of the 8-week treatment were compared to the values at the end of treatment, separately for each fish treatment. Comparisons were also performed by period since there were indications for period effect. Two-paired-samples The flow diagram of the study is shown in No significant differences were found for anthropometric indices, standard biochemical markers, circulating haemostatic markers or ex vivo platelet aggregation markers between the CF/EF and the EF/CF groups at the beginning of the study except that heart rate (pulses/min), fasting serum insulin concentration (pmol/L) and HOMA-IR were higher in the CF/EF group compared to the EF/CF group . FurtherParticipants’ compliance to the study protocol was high (100%), based on self-reporting, and this was confirmed by the RBC membrane content (%) in EPA and DHA, which was found to be higher at the end of each treatment period compared to the content at the beginning. Similar results were obtained for the consumption of the CF, as well as for the consumption of the EF during both treatment periods .p = 0.003), energy intake (p < 0.001), total and LDL-cholesterol , sP-selectin (p < 0.001), EC50ADP and EC50PAF . In particular, LDL-cholesterol was increased in the first period, whereas energy intake was decreased in the second period, regardless of treatment (data not shown).No significant sequence or carry-over effects were found for the changes in any of the primary outcomes or other outcome measures (data not shown). In contrast, there was a period  effect on some of the primary outcomes and other outcome measures. Specifically, the period of treatment affected BMI  for a period of eight weeks affected platelet aggregation and circulating haemostatic markers among apparently healthy adults. However, these effects were mostly period (season)-specific and did not differentiate between gilthead sea bream fed the olive pomace-enriched fish diet and gilthead sea bream fed the conventional fish diet.50 value of PAF in the second intervention period, whereas a non-significant increase was also found for the EC50 value of PAF after consumption of the enriched fish during the same period. In contrast, human platelets were more responsive to ADP after either fish consumption in the first period only, whereas no significant changes were observed in the second period. Thrombin-induced platelet aggregation was not significantly affected by either treatment. Previously, Mori and colleagues found that fish consumption significantly reduced platelet aggregation response to PAF stimulation only as part of a low-fat diet (30% of daily energy intake) [p > 0.4 for all). Lastly, it is now well established that other lipid constituents in fish and fish oils have biological activities which are not limited to their superior incorporation to plasma lipoproteins and cell membranes, and the AA/eicosanoid-related pathway [in vitro. In addition, fish polar lipids added in a cholesterol-rich diet and fed to rabbits for 45 days were shown to have anti-PAF and antithrombotic properties [With respect to platelet aggregation, consumption of the conventional fish increased the EC intake) . This fi intake) . Even th intake) . In addi intake) ,35 since pathway . Polar loperties .With respect to the circulating haemostatic markers, plasma PAI-1 concentration significantly increased after treatment with the conventional fish in the first period. Plasma sP-selectin concentration significantly increased after consumption of the conventional fish in the second period and tended to increase after consumption of the enriched fish in the second period as well. As in our study, plasma concentrations of soluble markers of platelet activation, CD40 ligand and P-selectin, were not significantly affected by consumption of 500 g of fatty fish per week for four weeks among healthy adults . PreviouOverall, our findings are in agreement with the findings from the few previously published studies which investigated the effects of differently fed farmed fish on CVD related biomarkers, mostly blood lipid indices. We found no significant changes in fasting serum concentration of triglycerides or HDL-cholesterol. There was a trend towards an increase in fasting serum total cholesterol and LDL-cholesterol after consumption of the conventional fish in the first period, and an increase in both total and LDL-cholesterol after consumption of the enriched fish in the first period, as well (data not shown). In two dietary intervention trials, apparently healthy adults randomly received 150 g per day marine trout fillet fed a 100% marine-based meal or a 100% vegetable-based meal for eight weeks  or approIn the present study, fasting serum concentrations of glucose and insulin, as well as HOMA-IR were not significantly affected by consumption of the conventionally fed gilthead sea bream in the first or the second period. Similarly, no significant changes were observed after consumption of the gilthead sea bream fed the olive pomace-enriched diet in the first period (data not shown). Our results are in accordance with previously published intervention studies where even high intake of fish oil supplement or frequent consumption of fatty fish did not improve parameters of glucose metabolism ,41. WithIn the study presented herein, we hypothesized that the consumption of fillets from fish fed with a diet where fish oil in the grow-out feed of gilthead sea bream was partly replaced with olive pomace (OP diet), would have a greater impact on PAF- induced platelet aggregation and haemostatic markers compared to consumption of fillets from fish fed with the fish oil-diet, based on the findings of previous experiments on the inhibition of the PAF-induced aggregation. Specifically, the OP diet contained polar lipid extracts of olive pomace that were found to inhibit PAF-induced washed rabbit platelet aggregation . Additio50 value of PAF after treatment with respect to before treatment and, thus, the study may not be sufficiently powered for the other primary outcomes evaluated. A second limitation is the seasonal effect on anthropometric and biochemical indices that may have an impact or conceal the differences in the primary outcomes. Certain strengths were also identified in the present study. In the absence of previously published data with respect to the effects of consumption of differently fed farmed gilthead sea bream on platelet aggregation and circulating haemostatic markers among apparently healthy adults, the cross-over design of the study is an important strength since participant serves as their own control. In addition, the absence of differences between the two sequence groups at the beginning of the study as well as the absence of carry-over effects with respect to the primary outcomes adds to the value of the study findings. Finally, participants’ compliance with fish consumption was also based on the findings from the fatty acid analysis of the red blood cell membranes.We must point out certain limitations and strengths of the present study to facilitate the interpretation of the findings. The first limitation is concerned with the determination of sample size. Power calculation was based on the ECIn conclusion, consumption of fillets from gilthead sea bream fed with a diet where fish oil in the grow-out fish feed was partly replaced with olive pomace did not have a greater impact on platelet aggregation and circulating haemostatic markers compared to consumption of fillets from fish fed the typical fish-oil diet among apparently healthy adults. It should be pointed out that the intervention was performed in Greek population where widespread consumption of olive oil exists and for that reason the conclusion cannot be generalized to different populations. Clinical trials with larger number of participants, and with a longer-term intervention evaluating various markers of cardiovascular disease could distinguish between the health effects of a conventionally fed gilthead sea bream and those of a differently fed gilthead sea bream. Future research could also focus on the enrichment of the fish feed with the isolated olive pomace polar lipid fraction, containing the bioactive specific PAF-receptor antagonists, and subsequent dietary intervention studies should evaluate the effect of the enriched fish on coagulation markers in humans.